Methods and systems for noninvasive and localized brain liquid biopsy using focused ultrasound

ABSTRACT

Among the various aspects of the present disclosure is the provision of a noninvasive and localized brain liquid biopsy using focused ultrasound. Briefly, therefore, the present disclosure is directed to methods and systems to identify brain lesion or tumor characteristics without the need for a solid brain biopsy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 62/662,013 filed on 24 Apr. 2018, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

MATERIAL INCORPORATED-BY-REFERENCE

The Sequence Listing, which is a part of the present disclosure,includes a computer readable form comprising nucleotide and/or aminoacid sequences of the present invention. The subject matter of theSequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to methods and systems fornon-invasive brain biopsies.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision ofa non-invasive and localized brain liquid biopsy using a focusedultrasound system. Briefly, therefore, the present disclosure isdirected to improved methods to identify brain tumor or lesioncharacteristics without the need for a solid brain biopsy.

One aspect of the present disclosure provides for methods fornon-invasively obtaining a biopsy of a brain tumor or lesion usinghigh-resolution MRI guided focused ultrasound and blood based genetictesting or biomarker detection.

Another aspect of the present disclosure provides for methods forbiopsying or diagnosing a brain tumor or lesion, non-invasively, usingMRI guided ultrasound and genetic testing or biomarker detection.

Another aspect of the present disclosure provides for methods fordisrupting brain tumor cells to identify genetic information or usingsequencing to identify or diagnose a tumor or lesion.

Another aspect of the present disclosure provides for methods for usingfocused ultrasound to release genetic information across the blood brainbarrier (BBB) and analyzing the genetic information.

Another aspect of the present disclosure provides for a method ofnon-invasively releasing biomarkers from a brain or a brain regionacross a blood brain barrier (BBB) of a subject.

In some embodiments, the method comprises applying a focused ultrasound(FUS) to the brain or the brain region, wherein the FUS is applied for aperiod of time and at an acoustic pressure sufficient to disrupt the BBBand release a detectable quantity of a biomarker across the BBB;obtaining a biological sample from the subject after applying the FUS toa subject brain or a subject brain region; or detecting a biomarker inthe biological sample.

In some embodiments, the biological sample is a biological fluidselected from the group consisting of: blood, cerebral spinal fluid(CSF), interstitial fluid (ISF), serum, and plasma.

In some embodiments, the period of time sufficient to disrupt the bloodbrain barrier (BBB) and release a detectable quantity of a biomarkeracross the BBB is between about 1 minute and about 4 minutes.

In some embodiments, the acoustic pressure sufficient to disrupt theblood brain barrier (BBB) and release a detectable quantity of abiomarker across the BBB is between about 0.1 MPa and about 10 MPa.

In some embodiments, the biomarker comprises genetic material.

In some embodiments, the genetic material is selected from cell-freeRNA, cell-free DNA, mRNA, circulating tumor DNA (ctDNA), or plasma DNAconcentration.

In some embodiments, the biomarker is selected from D-2-hydroxyglutarate(D2HG) or IDH1(R132H) mutation.

In some embodiments, the method comprises: scanning a subject head usinga magnetic resonance imaging (MRI) scanner and stereotacticallycoregistering the subject head and identifying a region to be targetedwith the FUS.

In some embodiments, the method comprises assessing the effectiveness ofthe BBB disruption or release of biomarkers comprising measuring MRIcontrast before and after FUS, wherein an increase in MRI contrast afterFUS compared to the MRI contrast before FUS indicates successful releaseof biomarker from the brain or brain region.

In some embodiments, detecting the biomarker in a biological samplecomprises genetic testing or sequencing.

In some embodiments, the method comprises extracting cell-free orexosomic DNA or RNA from the biological sample.

In some embodiments, the method comprises detecting somatic mutations inthe DNA or RNA using a targeted ultra-deep sequencing technologyselected from the group consisting of: ddPCR, AmpliSeq, and HaloPlexsequencing.

In some embodiments, the method comprises comparing a level of abiomarker in the biological sample after administering the FUS to abiological sample obtained from the subject before FUS or of a matchedcontrol sample or standard sample.

In some embodiments, the brain or brain region comprises a tumor,lesion, or suspected tumor.

In some embodiments, the subject has or is suspected of having a centralnervous system cancer or tumor; a brain tumor, a brain lesion, aneurological disease, disorder, or condition, or a neurodegenerativedisease disorder, or condition.

In some embodiments, the FUS is applied for a period of time and at apressure sufficient to rupture cells at the region to releasebiomarkers.

In some embodiments, the method comprises administering microbubbles toa subject in an amount sufficient to disrupt the BBB upon application ofFUS. In some embodiments, the method comprises providing an acousticsensor; or detecting an acoustic signal, wherein the sensor is capableof measuring or monitoring cavitational acoustic emissions.

Another aspect of the present disclosure provides for a system suitablefor use in delivering focused ultrasound (FUS) to a brain or a region ofa brain of a subject. In some embodiments, the system comprises a devicecomprising a plurality of modular pieces configured to deliverultrasound to the brain or a brain region. In some embodiments, thesystem comprises an ultrasound transducer configured to emit a FUS beam.In some embodiments, the system comprises an acoustic lens configured tocontrol the FUS beam direction.

In some embodiments, the system is configured for incorporation into amagnetic resonance imaging (MRI) scanner, wherein the MRI scanner isconfigured to provide an MRI image for guiding or monitoring of the FUS.

In some embodiments, the system comprises a helmet configured to fitover a head of a subject.

In some embodiments, the helmet is demountably coupled to the ultrasoundtransducer; the ultrasound transducer is demountably coupled to theacoustic lens; the ultrasound transducer is a flat ultrasoundtransducer; the acoustic lens is a 3D printed acoustic lens; or theacoustic lens is a convex lens.

In some embodiments, the system is suitable for use in parallel with aplurality of the systems.

In some embodiments, the system comprises a sensor capable of measuringor monitoring cavitational acoustic emissions.

In some embodiments, the ultrasound transducer is configured to emit alow acoustic pressure of less than about 10 MPa.

In some embodiments, the system comprises one to eight transducers.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1A-FIG. 1F. Experimental method. (A) Schematic illustration of thefocused ultrasound (FUS) experiment setup for the treatment of U87tumor-bearing mice. (B) Bioluminescence image of the orthotopic mousemodel with the green fluorescence image of the mouse brain shown on theright. (C) Schematic illustration of the MR-guided focused ultrasound(MRgFUS) system for the treatment of GL261 tumor-bearing mice. (D)Representative contrast-enhanced MR images acquired before and after FUStreatment. The enhanced accumulation of the MR contrast agents in thetumor region verified accurate tumor targeting by the FUS. (E) Diagramof FUS pulses. (F) Illustration of the experimental timeline.

FIG. 2A-FIG. 2C. Comparison of the circulating eGFP mRNA expression inthe control and treated U87 mice. (A) Amplification curves [log₂(ΔRn)]of circulating eGFP mRNA in the control (n=3) and treated mice (n=6) fortwo primer pairs, eGFP A and eGFP B. ΔRn is the fluorescence intensityof eGFP mRNA minus the baseline. Comparison of the relative expressionlevels (2^(−ΔC) _(T)) of (B) eGFP A and (C) eGFP B in the control andtreated mice. *: p<0.05

FIG. 3A-FIG. 3B. Comparison of the circulating eGFP mRNA expression inthe control and treated GL261 mice. Comparison of the expression levels(2^(−ΔC) _(T)) of (A) eGFP A and (B) eGFP B in the control group andthree treatment groups with different acoustic pressures: 1.48 MPa, 2.10MPa, and 3.34 MPa. All the measured data points as well as their meanand standard deviation are shown for each group. The circulating mRNAlevels of eGFP A and eGFP B were significantly higher in the FUS-treatedgroups compared with the untreated control group (eGFP A: p=0.0045; eGFPB: p=0.0045). The expression levels of mice in the 1.48 MPa group wassignificantly higher than those of the other two groups for eGFP A andeGFP B (eGFP A: p=0.012; eGFP B: p=0.012).

FIG. 4A-FIG. 4D. Histological assessment of brain tissue from thecontrol and treated GL261 mice. H&E staining of the ex vivo tumor slicesobtained from the control mice (A) and mice treated with FUS at (B) 1.48MPa, (C) 2.10 MPa, and (D) 3.34 MPa, respectively. Hemorrhages wereobserved in all the FUS treated mice.

FIG. 5A-FIG. 5C. Experimental setup. (A) Illustration and (B) picture ofthe MR-guided FUS system used for the FUS-LBx treatment. A clinicalMR-guided FUS system that integrated a clinical MRI scanner with a FUSphased array was configured for small animal study by coupling with asmall animal adaptor (see Methods for more details). (C) MR image of afiber-optic hydrophone used for measuring the acoustic pressures. Thehydrophone was placed vertically in a water container that was on top ofthe small animal adapter. The FUS transducer's focus (red dot) wasaligned at the tip of the optical fiber for pressure measurements.

FIG. 6A-FIG. 6B. Effect of peak negative pressure on FUS-LBx yield.Comparison of the circulating eGFP mRNA level in the control micereceiving no treatment and FUS-treated mice at 0.59, 1.29, and 1.58 MPa.eGFP mRNA levels were quantified using quantitative polymerase chainreaction (qPCR) with (A) primer A and (B) primer B.

FIG. 7A-FIG. 7F. Effect of peak negative pressure on brain hemorrhage.(A-D) Representative images of H&E-stained sections from mice in thecontrol group, 0.59 MPa, 1.29 MPa, and 1.58 MPa groups, respectively.Boxes in the images on the top indicate the location where the highermagnification images (bottom) were acquired. (E) Illustration ofmicrohemorrhage quantification using the positive pixel detectionalgorithm (see Methods). Yellow lines highlight the detectedmicrohemorrhage. (C) Microhemorrhage density is significantly higher inmice treated with higher pressures (1.29 MPa and 1.58 MPa) compared tothe low pressure (0.59 MPa) and control mice.

FIG. 8A-FIG. 8E. Relationships between intratumoral and peritumoral MRimage contrast enhancements and microhemorrhage density and eGFP mRNAlevel. (A) Overview of MRI analysis workflow. Tumor MR contrastenhancement was calculated for intratumoral area and peritumoral area(defined as the brain region within 2 mm vicinity of the tumor). (B)Correlation analyses between intratumoral and peritumoral enhancementswith microhemorrhage density and plasma eGFP mRNA level in FUS-treatedmice (n=15) using primer A.

FIG. 9 is a photo of the transducer.

FIG. 10 is a photo of a mouse treated using the device.

FIG. 11 is a photo showing the successful drug delivery to the mousebrain, indicated by the leakage of the blue color.

FIG. 12 is an image of spatially precise drug delivery to thehippocampus, indicated by the enhanced fluorescence observed on theright side of the of the mouse hippocampus.

FIG. 13 is a series of images of an MRgFUS system design for a pig.

FIG. 14. Planning and targeting of image-guided focused ultrasoundliquid biopsy (FUS-LBx).

FIG. 15. Monitoring and feedback control using an US sensor to monitormicrobubble response to US.

FIG. 16. Outcome assessment with MRI.

FIG. 17. FUS-LBx in a large animal model (pig). GFAP:Glial fibrillaryacidic protein; MBP: Myelin basic protein; S100B: S100 calcium-bindingprotein B.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery thata “liquid brain biopsy” can provide similar levels of information as astandard brain biopsy. As described herein, it was demonstrated that thecombination of FUS and microbubbles allows for the detection oftumor-specific mRNA in the bloodstream that is otherwise undetectable.These presently disclosed findings established that FUS-mediated BBBdisruption could enhance brain-to-blood trafficking. FUS may offer anenabling technique for a noninvasive and regionally-specific brain tumorliquid biopsy that can be utilized in personalized brain cancer patientcare.

In typical scenarios when a brain lesion is identified, or when apatient has a known brain tumor that has been previously treated, thereis a clinical indication to perform a surgical stereotactic biopsy. Thisentails making a hole in the skin and skull and placing a needle probeinto the lesion and aspirating a piece of tissue for histological andgenetic testing to achieve a diagnosis. Also of note, there are a numberof other lesions in the brain that can potentially requirebiopsy—lesions of unknown etiology, radiation necrosis, or known tumors,which require further clarification if genetic profile has changed, etc.This procedure can carry the surgical risks of bleeding, infection,non-diagnosis, or the requirements of general anesthesia.

This invention provides similar levels of information without therequirement for surgery. The general concept involves the followingprocess. A lesion in the brain is identified that requires pathologicdiagnosis. The lesion is imaged with MRI using high-resolution anatomicimaging (i.e., T1 and T2 sequences). The imaging is stereotacticallycoregistered to the patient's head using standard frameless navigationsystems. Targets for biopsy are then identified with the navigationsystem. Once done, a focused ultrasound source is then used to targetthe region. The patient is concurrently given ultrasound fluoridatedmicrobubbles (microbubbles can be optional). The focused ultrasound orcombination of the ultrasound and microbubbles can induce a disruptionof the blood brain barrier in the prospectively chosen target site. Analternative to the fluoridated microbubbles would be to simply havehigher power focused ultrasound that creates a local tissue injury anddisruption of the blood brain barrier. This US perturbation can alsolead to the rupture of the cells and the release of genetic materialinto the blood stream. Biological samples (e.g., blood) and, optionally,matched normal samples can then be taken from the patient andgenetically analyzed in order to identify biomarkers, such ascirculating somatic variants. These somatic variants can then beevaluated to determine the nature of the lesion in the brain.

The technology/process can involve the novel combination of focusedultrasound and advanced genetic screening. Specifically, the ultrasoundtechnique can be a simple, compact, and relatively inexpensive focusedultrasound system that is comprised of a single-element FUS transducer.The transducer can be coupled to a high-precision, fast-speedpositioning system to steer transducer focus mechanically. It is also anoption to use a focused ultrasound system comprising a phased arraytransducer with multiple elements for steering the transducer focalpoint electronically.

These techniques can be used in conjunction with either stereotacticnavigation in which the patient's brain imaging and lesion localizationis rigorously coregistered in physical geometric space. This can allowfor precise perturbation of the intended target. Once performed, bloodcan be withdrawn from the patient and cell-free or exosomic DNA and/orRNA can be extracted. The DNA and/or RNA can be evaluated for thepresence of somatic mutations using a targeted ultra-deep sequencingtechnology, examples of which include, but are not limited to, PCR,qPCR, ddPCR, AmpliSeq, or HaloPlex sequencing. The set of variants to bequeried can contain patient-specific variants (identified usingwhole-genome or whole-exome sequencing of a surgically-resected tumorand matched normal tissue from the same patient), a panel of variantsthat are commonly present in brain tumors or a combination of panelvariants and patient-specific variants. Optionally, genomic DNA frommatched normal samples from the same patient can be sequenced and usedto distinguish somatic variants from germline variants.

It is also important to note that while blood is the easiest and mostaccessible source of fluid to perform further genetic analysis, onecould also perform this same analysis on cerebrospinal fluid (CSF) orother biological samples (e.g., blood, plasma, serum, interstitial fluid(ISF)).

Blood Brain Barrier (BBB) Opening with Focused Ultrasound (FUS)

The blood-brain barrier (BBB) blocks large molecules (>400 Da) fromentering the central nervous system (CNS). Focused Ultrasound (FUS) isthe only available technique to non-invasively, locally, and transientlyopen the BBB. Previous preclinical research using small animal modelshas demonstrated this technique as a promising way to treat variousbrain disorders. FUS in combination with microbubbles can also be used.This technique has also been tested on non-human primates with success.Clinical Studies with FUS are actively investigated for treatment ofbrain tumor and Alzheimer's disease. Besides its clinical translationpotential, this technique has a broad application in preclinicalresearch. This technique can be used to deliver various agents to thebrain, such as chemo drugs, proteins, peptides, nanoparticles, genevectors, and stem cells. However, currently there is a lack of devicesthat are affordable, high throughput, and dedicated for small animalresearch. Here is disclosed a FUS-induced BBB opening device that meetsthese needs.

The BBB, as described herein, can also comprise a blood-tumor barrier.The blood-tumor barrier is less leaky than vessels outside the centralnervous system, but is leakier than a healthy BBB.

As described herein, the BBB can be opened for minutes, hours, or days,depending on the US energy administered to the subject. This can allowfor serial testing of biomarkers. It is preferred to have as short of anopen window as possible (e.g., on the order of minutes) because of thepossibility of infection or because the detectable biomarkers can have ashort half-life, are unstable, or are quickly cleared from thebloodstream. Because the half-life for many biomarkers is known, thetime point for collection is known as well (e.g., the time at which asample is taken form a subject can correlate to a time where thebiomarker has not yet been cleared from the subject's system).

Focused Ultrasound (FUS) System

Disclosed herein is a focused ultrasound (FUS) system for use indisruption of a blood brain barrier (BBB), releasing biomarkers from abrain lesion or tumor. After the subject is administered the ultrasound,the biomarkers can be detected in a biological sample of a subject.Conventional FUS systems being used to open up the BBB are being usedwith therapeutically-relevant acoustic pressures (e.g., higher peaknegative acoustic pressure (PNP) levels than used in typical diagnosticor imaging levels). For example, clinical FUS systems use relativelyhigh acoustic pressure. The FUS systems and methods as described hereincan use diagnostically-relevant acoustic pressures to open the bloodbrain barrier and release biomarkers from a brain lesion or suspectedbrain lesion.

As an example, the acoustic pressure for mice in the presently disclosedsystem can be about 0.6 MPa. As another example, the acoustic pressurefor a human in the presently disclosed system can be about 1.5 MPa.

Furthermore, conventional clinical systems use high energy and requirehigh precision for use in delivering treatment to a specific area, suchas a tumor or lesion. The FUS system described herein does not requirehigh energy or precision to disrupt the BBB and release biomarkers fromthe brain or a brain lesion. For example, a clinical FUS system cancomprise over 1,000 elements (a transducer array) that are individuallysteered. The system described herein only requires a single element (butup to about 8 can be useful) at diagnostically-relevant energyrequirements. This design decreases the cost, simplifies the design, canbe adapted for human or animal use, and is designed to be incorporatedinto any MRI scanner.

Described herein in also a method of monitoring cavitation (e.g.,microbubble behavior) (see e.g., Example 3). The FUS system can comprisea sensor that measures acoustic signal. This monitoring can inform ifthe acoustic pressure should be increased or decreased by measuring theacoustic signal.

As described herein, the focused ultrasound-enabled brain tumor liquidbiopsy (FUS-LBx) offers a technique that can provide noninvasive,spatially resolved, and temporally controlled brain liquid biopsies. FUShas emerged as a technology with the potential to noninvasively exertmechanical and thermal effects on the brain tissue. When coupled withintravenously injected microbubbles, low-intensity pulsed FUS-inducedmechanical effects can transiently and non-invasively open the BBBwithout causing any or substantial vascular or tissue damage. It wasdemonstrated (see e.g., Example 1) that FUS in combination withmicrobubbles could also be exploited to release biomarkers from braintumors in murine glioblastoma models. The approach described here, isdifferent from previously reported FUS-facilitated liquid biopsytechniques developed for in vivo biomarker release from cancers outsidethe brain. In one of those reports, pulsed high-intensity focusedultrasound (HIFU) with high acoustic pressures (ultrasound frequency=1.5MHz, peak compressional focal pressure=90 MPa, and peak rarefactionalfocal pressure=17 MPa) was used to induce histotripsy (i.e., a techniquefor mechanical tissue fractionation) in a rat model of prostate cancer,and this enhanced the release of cell-free tumor microRNA into the bloodcirculation. In another study, a chicken embryo tumor model was used toshow the feasibility of amplifying the release of extracellular vesiclesusing HIFU (ultrasound frequency=1.15 MHz and peak to peak pressure waswithin the range of 10-30 MPa) in combination with phase-changenanodroplets which changed to microbubbles upon HIFU sonication. In arecent study, two protein biomarkers were found to be significantlyincreased in the plasma of patients undergoing HIFU thermal ablation(ultrasound frequency=1.1 MHz and power of 100-200 W) of uterinefibroids. All these previous studies used HIFU to induce permanentmechanical or thermal disruption of the tumor to enhance the release oftumor biomarkers. The tissue damaging effect limits the application ofthese techniques as diagnostic tools in a sensitive organ such as thebrain. In contrast, the FUS-LBx technique disclosed herein useslow-intensity pulsed FUS, which has the potential advantage of enablingthe biomarker release without causing tissue damage. As shown in Example1, the acoustic pressures used were intentionally selected to berelatively high (1.52-3.53 MPa in mouse model) to increase the chance ofsuccess in releasing biomarkers. As expected, hemorrhage was found inthe relatively higher intensity FUS-targeted brain area.

Low-intensity focused ultrasound can be used with low acoustic pressures(e.g., less than about 10 MPa), ultrasound frequency less than about 1.5MHz, peak compressional focal pressure less than about 90 MPa, or peakrarefactional focal pressure less than about 17 MPa. Low-intensityfocused ultrasound can be used with a peak to peak pressure less thanabout 10 to 30 MPa; an ultrasound frequency less than 1.1 MHz; a powerof less than about 100 to 200 W; or peak negative acoustic pressure(PNP) levels of less than about 3.53 MPa.

Example 2 describes the effects of FUS acoustic pressure on the level oftumor biomarker release and the extent of associated hemorrhage in orderto optimize the FUS-LBx technique. Example 2 determined the optimalultrasonic pressure for FUS-LBx that can sufficiently increase plasmalevels of the tumor biomarkers while at the same time minimize the riskof hemorrhage in the brain. It was further explored whetherpost-sonication changes in tumor MR contrast enhancement can predictsuccessful biomarker release for the future development of image-guidedFUS-LBx technique.

Disclosed herein are systems comprising a FUS-induced BBB opening devicethat are affordable, high throughput, and can be easily translated forclinical use or dedicated for animal (e.g., pig) or small animal (e.g.,mouse) research. The transducer, as described herein can be aninexpensive flat ultrasound transducer coupled to a 3D printed acousticlens (or acoustic window). The cost of the transducer, as described inthe Examples, was about $80, while the FUS transducer commonly used inpreclinical research can cost over $2,000. Multiple transducers can beused simultaneously to achieve high throughput treatment of multipleanimals at the same time.

A wearable helmet can be used for non-invasive, targeted FUS sonicationof the human or animal brain while animals are awake. The helmet canhave a modular design, featuring easy removal and installation of theunit for targeting different brain regions.

The performance of the helmet in inducing BBB opening can be assessed byco-injecting Evan's blue with microbubbles to evaluate BBB permeabilityusing fluorescence imaging of ex vivo brain slices in animals.

The helmet and ultrasound transducer and wirings are lightweight atabout 6.6 g. The constraint design of the helmet can minimize the effectof mouse movement on targeting. The helmet system achieved localized BBBopening at the targeted brain location. The fluorescence intensity ofthe Evan's blue in the brains of awake mice was higher than that inanesthetized mice, suggesting that FUS-induced BBB opening was affectedby anesthesia.

The helmet design of the FUS device provides an innovative tool to studyFUS-induced BBB opening in subjects such as an awake mouse.

As described herein, the FUS system can use acoustic pressures betweenabout 0.1 MPa and about 10 MPa. For example, the acoustic pressure canbe about 0.1 MPa; about 0.2 MPa; about 0.3 MPa; about 0.4 MPa; about 0.5MPa; about 0.6 MPa; about 0.7 MPa; about 0.8 MPa; about 0.9 MPa; about 1MPa; about 1.1 MPa; about 1.2 MPa; about 1.3 MPa; about 1.4 MPa; about1.5 MPa; about 1.6 MPa; about 1.7 MPa; about 1.8 MPa; about 1.9 MPa;about 2 MPa; about 2.1 MPa; about 2.2 MPa; about 2.3 MPa; about 2.4 MPa;about 2.5 MPa; about 2.6 MPa; about 2.7 MPa; about 2.8 MPa; about 2.9MPa; about 3 MPa; about 3.1 MPa; about 3.2 MPa; about 3.3 MPa; about 3.4MPa; about 3.5 MPa; about 3.6 MPa; about 3.7 MPa; about 3.8 MPa; about3.9 MPa; about 4 MPa; about 4.1 MPa; about 4.2 MPa; about 4.3 MPa; about4.4 MPa; about 4.5 MPa; about 4.6 MPa; about 4.7 MPa; about 4.8 MPa;about 4.9 MPa; about 5 MPa; about 5.1 MPa; about 5.2 MPa; about 5.3 MPa;about 5.4 MPa; about 5.5 MPa; about 5.6 MPa; about 5.7 MPa; about 5.8MPa; about 5.9 MPa; about 6 MPa; about 6.1 MPa; about 6.2 MPa; about 6.3MPa; about 6.4 MPa; about 6.5 MPa; about 6.6 MPa; about 6.7 MPa; about6.8 MPa; about 6.9 MPa; about 7 MPa; about 7.1 MPa; about 7.2 MPa; about7.3 MPa; about 7.4 MPa; about 7.5 MPa; about 7.6 MPa; about 7.7 MPa;about 7.8 MPa; about 7.9 MPa; about 8 MPa; about 8.1 MPa; about 8.2 MPa;about 8.3 MPa; about 8.4 MPa; about 8.5 MPa; about 8.6 MPa; about 8.7MPa; about 8.8 MPa; about 8.9 MPa; about 9 MPa; about 9.1 MPa; about 9.2MPa; about 9.3 MPa; about 9.4 MPa; about 9.5 MPa; about 9.6 MPa; about9.7 MPa; about 9.8 MPa; about 9.9 MPa; or about 10 MPa. Recitation ofeach of these discrete values is understood to include ranges betweeneach value. Recitation of each range is understood to include discretevalues within the range.

As described herein, the FUS system can use a duty cycle between about0.1% and about 10%. For example, the duty cycle can be about 0.1%; about0.2%; about 0.3%; about 0.4%; about 0.5%; about 0.6%; about 0.7%; about0.8%; about 0.9%; about 1%; about 1.1%; about 1.2%; about 1.3%; about1.4%; about 1.5%; about 1.6%; about 1.7%; about 1.8%; about 1.9%; about2%; about 2.1%; about 2.2%; about 2.3%; about 2.4%; about 2.5%; about2.6%; about 2.7%; about 2.8%; about 2.9%; about 3%; about 3.1%; about3.2%; about 3.3%; about 3.4%; about 3.5%; about 3.6%; about 3.7%; about3.8%; about 3.9%; about 4%; about 4.1%; about 4.2%; about 4.3%; about4.4%; about 4.5%; about 4.6%; about 4.7%; about 4.8%; about 4.9%; about5%; about 5.1%; about 5.2%; about 5.3%; about 5.4%; about 5.5%; about5.6%; about 5.7%; about 5.8%; about 5.9%; about 6%; about 6.1%; about6.2%; about 6.3%; about 6.4%; about 6.5%; about 6.6%; about 6.7%; about6.8%; about 6.9%; about 7%; about 7.1%; about 7.2%; about 7.3%; about7.4%; about 7.5%; about 7.6%; about 7.7%; about 7.8%; about 7.9%; about8%; about 8.1%; about 8.2%; about 8.3%; about 8.4%; about 8.5%; about8.6%; about 8.7%; about 8.8%; about 8.9%; about 9%; about 9.1%; about9.2%; about 9.3%; about 9.4%; about 9.5%; about 9.6%; about 9.7%; about9.8%; about 9.9%; or about 10%. Recitation of each of these discretevalues is understood to include ranges between each value. Recitation ofeach range is understood to include discrete values within the range.

As described herein, the FUS system can use a pulse repetition frequencybetween about 0.1 Hz and about 10 Hz. For example, the pulse repetitionfrequency can be about 0.1 Hz; about 0.2 Hz; about 0.3 Hz; about 0.4 Hz;about 0.5 Hz; about 0.6 Hz; about 0.7 Hz; about 0.8 Hz; about 0.9 Hz;about 1 Hz; about 1.1 Hz; about 1.2 Hz; about 1.3 Hz; about 1.4 Hz;about 1.5 Hz; about 1.6 Hz; about 1.7 Hz; about 1.8 Hz; about 1.9 Hz;about 2 Hz; about 2.1 Hz; about 2.2 Hz; about 2.3 Hz; about 2.4 Hz;about 2.5 Hz; about 2.6 Hz; about 2.7 Hz; about 2.8 Hz; about 2.9 Hz;about 3 Hz; about 3.1 Hz; about 3.2 Hz; about 3.3 Hz; about 3.4 Hz;about 3.5 Hz; about 3.6 Hz; about 3.7 Hz; about 3.8 Hz; about 3.9 Hz;about 4 Hz; about 4.1 Hz; about 4.2 Hz; about 4.3 Hz; about 4.4 Hz;about 4.5 Hz; about 4.6 Hz; about 4.7 Hz; about 4.8 Hz; about 4.9 Hz;about 5 Hz; about 5.1 Hz; about 5.2 Hz; about 5.3 Hz; about 5.4 Hz;about 5.5 Hz; about 5.6 Hz; about 5.7 Hz; about 5.8 Hz; about 5.9 Hz;about 6 Hz; about 6.1 Hz; about 6.2 Hz; about 6.3 Hz; about 6.4 Hz;about 6.5 Hz; about 6.6 Hz; about 6.7 Hz; about 6.8 Hz; about 6.9 Hz;about 7 Hz; about 7.1 Hz; about 7.2 Hz; about 7.3 Hz; about 7.4 Hz;about 7.5 Hz; about 7.6 Hz; about 7.7 Hz; about 7.8 Hz; about 7.9 Hz;about 8 Hz; about 8.1 Hz; about 8.2 Hz; about 8.3 Hz; about 8.4 Hz;about 8.5 Hz; about 8.6 Hz; about 8.7 Hz; about 8.8 Hz; about 8.9 Hz;about 9 Hz; about 9.1 Hz; about 9.2 Hz; about 9.3 Hz; about 9.4 Hz;about 9.5 Hz; about 9.6 Hz; about 9.7 Hz; about 9.8 Hz; about 9.9 Hz; orabout 10 Hz. Recitation of each of these discrete values is understoodto include ranges between each value. Recitation of each range isunderstood to include discrete values within the range.

As described herein, the FUS system can use an exposure duration betweenabout 1 second and about 20 minutes. For example, the exposure durationcan be about 30 s; about 1 min; about 2 min; about 3 min; about 4 min;about 5 min; about 6 min; about 7 min; about 8 min; about 9 min; about10 min; about 11 min; about 12 min; about 13 min; about 14 min; about 15min; about 16 min; about 17 min; about 18 min; about 19 min; or about 20min. Recitation of each of these discrete values is understood toinclude ranges between each value. Recitation of each range isunderstood to include discrete values within the range.

As described herein, a biological sample can be collected at any timeprior to or after FUS. For example, the sample collection can correspondwith a time less than the half-life of the biomarker to be tested. Asanother example, biological samples can be serially collected at varioustime points before or after FUS. As another example, the biologicalsample can be collected within 10 minutes to about 2 days after FUSexposure or administration. For example, the biological sample can becollected post-FUS at about 1 min, about 2 min, about 3 min; about 4min; about 5 min; about 6 min; about 7 min; about 8 min; about 9 min;about 10 min; about 11 min; about 12 min; about 13 min; about 14 min;about 15 min; about 16 min; about 17 min; about 18 min; about 19 min; orabout 20 min. Recitation of each of these discrete values is understoodto include ranges between each value. Recitation of each range isunderstood to include discrete values within the range.

As described herein, the system can be used for any subject with acentral nervous system and a blood brain barrier. For example, thesubject can be an animal subject, including a mammal, such as horses,cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guineapigs, and chickens, and humans. As another example, the subject can be ahuman subject.

Biological Sample

The methods and systems designed herein can release biomarkers orgenetic material from a brain into a biological sample, such as abiological fluid. The biological fluid or biological sample can be wholeblood, blood, plasma, serum, cerebral spinal fluid (CSF), orinterstitial fluid (ISF).

Microbubbles

The methods described herein can comprise the use of microbubbles.Microbubbles are widely used for the heart and liver. Microbubbles arecommercially available (e.g., Definity®). Microbubbles can be used withFUS to provide lower levels of US intensity with similar results tohigh-intensity US with less potential side effects or damage. Use ofmicrobubbles, generally, is well known; see e.g. Blomley et al. (2001)“Science, medicine, and the future: Microbubble contrast agents: A newera in ultrasound” BMJ 322 (7296): 1222-5; Sirsi et al. 2009 Bubble SciEng Technol 1(1-2): 3-17. Except as otherwise noted herein, therefore,the process of the present disclosure can be carried out in accordancewith such processes.

Microbubbles can be administered by methods currently known in the art.As described herein, the microbubbles are administered according to theclinical dose currently used in treatments (e.g., for Definity®microbubbles, 1.3 mL from a diluted vial is diluted with 8.7 mL saline).

Microbubbles are small, gas-filled bubbles, which can be between 0.5 μmand 10 μm in diameter. Microbubbles are widely used as contrast agentsin medical imaging and as carriers for targeted drug delivery. The coreof the microbubble is a gas, which is surrounded by a shell that can becomposed of polymers, lipids, lipopolymers, proteins, surfactants, or acombination of these.

Microbubbles can be injected intravenously, a process that researchershave shown is safe compared to the use of conventional contrast agentsin techniques such as magnetic resonance imaging and radiography.

Microbubbles resonate in an ultrasound beam, contracting and expandingas pressure changes occur in the ultrasound wave. Microbubbles resonateparticularly vigorously at the high frequencies used in ultrasound scans(they reflect these strong waves significantly more effectively thanbody tissues do). Since they are approximately the same size as redblood cells, they exhibit similar rheology in blood vessels and are usedto measure blood flow in organs and tumors.

Conventionally, microbubbles have been used for drug delivery in thetreatment of cancer. More recently, microbubbles have been used formolecular imaging and targeted gene delivery. It is believed that thisis the first reporting of microbubbles being used to facilitate therelease of biomarkers or genetic material across the blood brain barrier(BBB).

Microbubbles can be any microbubbles known in the art. For example, themicrobubbles can be commercially available microbubbles, such asDefinity® microbubbles or phase-change nanodroplets. For example, GEHealthcare makes Optison, a Food and Drug Administration (FDA)-approvedmicrobubble. Optison has an albumin shell and octafluoropropane gascore. Lantheus makes Definity®, which is a FDA-approved microbubble. InDefinity®, perflutren lipid microspheres are composed ofoctafluoropropane encapsulated in an outer lipid shell. Bracco makesLumason (SonoVue in other countries) which is also FDA-approved. Lumasonis a sulfur hexafluoride containing lipid-encapsulated microbubble.

Microbubbles can be used as described herein, but the use ofmicrobubbles is not required to achieve the opening of the BBB andrelease of biomarkers or genetic information from a brain lesion ortumor.

Biomarker Detection, Genetic Testing, and Genetic Sequencing

Described herein are systems and methods for releasing biomarkers orgenetic material from a brain into a biological sample for detection.Detection of these biomarkers can be used to diagnose or treat a subjector to formulate a personalized treatment plan. The presence or absenceof such biomarkers or genetic material has implications on clinicaldecision-making in standard clinical care.

Described herein is the use of qPCR to detect mRNA, but any method ofdetecting genetic material or biomarkers known in the art can be used.It was also shown that brain-specific biomarkers were released followingFUS to the brain (see e.g., Example 3).

The disclosed systems and methods can be used to detect already knownbiomarkers for a disease or discover new biomarkers in subjectsdiagnosed with a disease.

Biomarkers, such as RNA or DNA, can have a short half-life. As such, theperiod of time for the biological sample collection can be from about 1minute to about 5 minutes or more after FUS is applied to the brain or aportion of the brain. For example, a sample can be collected before FUS,during FUS, or after FUS. As another example, the time after FUSapplication to collect a biological sample can be about 1 min, about 2min; about 3 min; about 4 min; about 5 min; about 6 min; about 7 min;about 8 min; about 9 min; about 10 min; about 11 min; about 12 min;about 13 min; about 14 min; about 15 min; about 16 min; about 17 min;about 18 min; about 19 min; or about 20 min. A biological sample can becollected any time before the BBB closes. Recitation of each of thesediscrete values is understood to include ranges between each value.Recitation of each range is understood to include discrete values withinthe range.

A biological sample can be collected before FUS and after FUS to measurethe biomarkers. Biomarkers can be detected to monitor a course oftreatment or to monitor disease progression.

Widely-validated biomarkers that can be detected in a biological sampleafter FUS treatment can include (i) MGMT promoter methylation as aprognostic and predictive marker in glioblastoma; (ii) co-deletion of 1pand 19q differentiating oligodendrogliomas from astrocytomas; (iii)IDH1/2 mutations; or (iv) select pathway-associated mutations.

Biomarkers detected (with corresponding diseases presently known to berelevant for these biomarkers) can be: co-deletion 1p/19q(Oligodendrogliomas); MGMT promoter methylation (Glioblastomas,anaplastic astrocytomas); IDH 1/2 mutation (Oligo- and astrocytomas WHOgrade II and Ill, secondary GBM); EGFRvlII (Glioblastomas); TERTmutation (Gliomas); ATRX mutations (Gliomas); TP53 mutations(Astrocytomas); BRAF V600 mutation (PXA, pilocytic astrocytomas,gangliogliomas); BRAF/KIAA1549 fusion (Pilocytic astrocytomas); H3.3histones (Pediatric HGG); SHH pathway mutations (such as MYCNamplification, GLI2 amplification, or TP53 mutations)(Medulloblastomas); WNT pathway mutations (Medulloblastomas); or MYC(Medulloblastomas).

Detecting biomarkers of brain tumors in biological samples is well knownin the art (see e.g., Staedtke et al. (2016) “Actionable MolecularBiomarkers in Primary Brain Tumors” Trends in Cancer 2(7) 338-349).Except as otherwise noted herein, therefore, the process of detectingbiomarkers of the present disclosure can be carried out in accordancewith such processes.

Detecting biomarkers (e.g., DNA, RNA, cell-free RNA, cell-free DNA) ofneurodegenerative disease in biological samples is well known in the art(see e.g., Jeromin et al. (2017) Adv Neurobiol 15 491-528; Beach 217Neurol Ther. 6(Supp 1)5-13). Except as otherwise noted herein,therefore, the process of detecting biomarkers of the present disclosurecan be carried out in accordance with such processes.

As an example, the presently disclosed systems and methods can be usedfor detecting or discovering biomarkers for Alzheimer's disease (AD)(e.g., AR, tau, phosphorylated tau, other neuronal proteins),Parkinson's disease (PD) (e.g., alpha-synuclein), or amyotrophic lateralsclerosis (ALS).

Detecting biomarkers of neurological or neurodegenerative diseases,disorders, or conditions in biological samples are well known in the art(see e.g., Smith 2017 Biomarkers on the brain: Putting biomarkerstogether for a better understanding of the nervous system, Science,special technology feature; Jain 2015 Biomarkers in Neurology, MedLink).Except as otherwise noted herein, therefore, the process of the presentdisclosure can be carried out in accordance with such processes.

Detection and discovery of brain biomarkers can track diseaseprogression over time and correlate with known clinical measures; detectthe effect or efficacy of a drug; or serve as a surrogate endpoint inclinical trial.

Liquid Brain Biopsy

Described herein are methods and systems for blood-based liquid biopsiesof the brain. The liquid brain biopsy can be used to detect geneticmaterial from the brain from various central nervous system (CNS cancersor tumors or neurological or neurodegenerative diseases, disorders, orconditions). The disclosed methods and systems can reach lesionspreviously unreachable by traditional biopsy techniques. Furthermore,the disclosed systems and methods can be performed repeatedly for use inmonitoring the lesion, treatment response, or treatment efficacy. Thedisclosed systems and methods can also be useful for diagnosing lesionsor detecting biomarkers in areas of the brain or CNS that are typicallynot available for biopsy or surgery, such as the brain stem.

Previous methods for FUS on the brain focused on drug-delivery (“one-waytrafficking”). Here, it was discovered that FUS-mediated BBB disruptionenhances “two-way trafficking” between brain and blood.

Central nervous system (CNS) tumors are significant causes of cancermorbidity and mortality, especially in children and young adults wherethey account for ˜20-30% of cancer deaths. Noninvasive neuroimagingmodalities (e.g., magnetic resonance imaging (MRI), and computerizedtomography (CT)) are used to evaluate tumor lesions, but observedchanges, especially after treatment, can be difficult to interpret.Surgical resection or stereotactic biopsy is typically performed forhistologic confirmation and increasingly for genetic profiling. However,tissue biopsy requires brain surgery and can be associated with adverseeffects such as hemorrhage and infection. Repeated tumor biopsies thatmay be needed for tracking tumor evolution, treatment response, andtumor recurrence are often not feasible. Furthermore, tissue biopsiesmay be challenging when tumors are located at difficult locations orpatients are too ill to tolerate invasive procedures.

Noninvasive blood-based liquid biopsies are a rapidly emerging strategyto provide genetic tumor profiling that can be used for treatmentselection, treatment monitoring, residual disease detection, andasymptomatic individual screening. Some success has been achieved inintegrating blood-based liquid biopsy into the routine clinicaldiagnostics. However, limited progress has been made for brain tumors.Although several groups have reported biomarker detection in thecerebrospinal fluid (CSF) of patients with brain tumors, obtaining CSFvia lumbar puncture remains an invasive procedure and can be unsafe insome settings. Blood-based liquid biopsies are noninvasive, but thereremain multiple hurdles for their application in the brain. First, theblood-brain barrier (BBB) restricts the release of large molecules suchas DNA and RNA from the tumor to the peripheral circulation. Even thoughpartial BBB disruption is a core feature of glioblastoma (the mostcommon type of high-grade glioma) at the late stage of the tumors, theBBB remains intact in the early stage and the low-grade diffuse gliomas.Second, brain tumors, especially glioblastomas, are heterogeneous withspatially distinct molecular profiling. Localization of tumor areas thatharbor specific mutations is not possible using conventional methods ofliquid biopsy, as these methods are inherently spatially agnostic.Third, many tumor markers, such as some cell-free RNAs and DNAs, haveshort half-lives in blood. Detection of these biomarkers can be enhancedby stimulating their release from the tumor to the circulation andprecisely controlling the blood-collection time to be shorter than theirlifetimes in the blood. Therefore, the disclosed noninvasive, spatiallyresolved, and temporally controlled liquid biopsy technique is in greatneed to improve the clinical care of patients with any neurological orneurodegenerative disease, disorder, or condition (e.g., CNS tumors).

Central Nervous System (CNS) Cancer/Tumors

The presently disclosed methods can be used to detect or discoverbiomarkers for central nervous system (CNS) cancers or tumors. CNScancers can include a brain or spinal cord cancer or tumor.

The brain or spinal cord cancer or tumor detected can be acousticneuroma; astrocytoma; atypical teratoid rhaboid tumor (ATRT); brain stemglioma; chordoma; chondrosarcoma; choroid plexus; CNS lymphoma;craniopharyngioma; cysts; ependymoma; ganglioglioma; germ cell tumor;glioblastoma (GBM); glioma; hemangioma; juvenile pilocytic astrocytoma(JPA); lipoma; lymphoma; medulloblastoma; meningioma; metastatic braintumor; neurilemmomas; neurofibroma; neuronal & mixed neuronal-glialtumors; non-hodgkin lymphoma; oligoastrocytoma; oligodendroglioma; opticnerve glioma; pineal tumor; pituitary tumor; primitive neuroectodermal(PNET); rhabdoid tumor; or schwannoma.

The astrocytoma can be a grade I pilocytic astrocytoma, a gradeII—low-grade astrocytoma, a grade III anaplastic astrocytoma, a grade IVglioblastoma (GBM), or a juvenile pilocytic astrocytoma.

The glioma can be a brain stem glioma, an ependymoma, a mixed glioma, anoptic nerve glioma, or a subependymoma.

Depending on the marker, treatment of CNS cancers or tumors can comprisethe administration of PCV, temozolomide, IDH1/2inhibitors,Immunotherapeutic approaches (e.g., vaccines, CAR T-cells), BRAFinhibitors, MEK inhibitors, Epigenetic inhibitors, JMJD3 inhibitor, SMOinhibitors, reduced dose of RT, chemotherapy or combination, Gemcitabineand pemetrexed, or BET bromodomain inhibitors (see e.g., Staedtke et al.(2016) “Actionable Molecular Biomarkers in Primary Brain Tumors” Trendsin Cancer 2(7) 338-349).

Neurological or Neurodegenerative Diseases, Disorders, or Conditions

These disclosed methods and system can also be used to detect ordiscover biomarkers for other neurological disease states. For example,the methods and systems can be used in subjects having or suspected ofhaving a neurological disease, disorder, or condition such as Abulia;Agraphia; Alcoholism; Alexia; Alien hand syndrome; Allan-Herndon-Dudleysyndrome; Alternating hemiplegia of childhood; Alzheimer's disease;Amaurosis fugax; Amnesia; Amyotrophic lateral sclerosis (ALS); Aneurysm;Angelman syndrome; Anosognosia; Aphasia; Apraxia; Arachnoiditis;Arnold-Chiari malformation; Asomatognosia; Asperger syndrome; Ataxia;Attention deficit hyperactivity disorder; ATR-16 syndrome; Auditoryprocessing disorder; Autism spectrum; Behcets disease; Bipolar disorder;Bell's palsy; Brachial plexus injury; Brain damage; Brain injury; Braintumor; Brody myopathy; Canavan disease; Capgras delusion; Carpal tunnelsyndrome; Causalgia; Central pain syndrome; Central pontinemyelinolysis; Centronuclear myopathy; Cephalic disorder; Cerebralaneurysm; Cerebral arteriosclerosis; Cerebral atrophy; Cerebralautosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL); Cerebraldysgenesis-neuropathy-ichthyosis-keratoderma syndrome (CEDNIK syndrome);Cerebral gigantism; Cerebral palsy; Cerebral vasculitis; Cervical spinalstenosis; Charcot-Marie-Tooth disease; Chiari malformation; Chorea;Chronic fatigue syndrome; Chronic inflammatory demyelinatingpolyneuropathy (CIDP); Chronic pain; Cockayne syndrome; Coffin-Lowrysyndrome; Coma; Complex regional pain syndrome; Compression neuropathy;Congenital facial diplegia; Corticobasal degeneration; Cranialarteritis; Craniosynostosis; Creutzfeldt-Jakob disease; Cumulativetrauma disorders; Cushing's syndrome; Cyclothymic disorder; CyclicVomiting Syndrome (CVS); Cytomegalic inclusion body disease (CIBD);Cytomegalovirus Infection; Dandy-Walker syndrome; Dawson disease; DeMorsier's syndrome; Dejerine-Klumpke palsy; Dejerine-Sottas disease;Delayed sleep phase syndrome; Dementia; Dermatomyositis; Developmentalcoordination disorder; Diabetic neuropathy; Diffuse sclerosis; Diplopia;Disorders of consciousness; Down syndrome; Dravet syndrome; Duchennemuscular dystrophy; Dysarthria; Dysautonomia; Dyscalculia; Dysgraphia;Dyskinesia; Dyslexia; Dystonia; Empty sella syndrome; Encephalitis;Encephalocele; Encephalotrigeminal angiomatosis; Encopresis; Enuresis;Epilepsy; Epilepsy-intellectual disability in females; Erb's palsy;Erythromelalgia; Essential tremor; Exploding head syndrome; Fabry'sdisease; Fahr's syndrome; Fainting; Familial spastic paralysis; Febrileseizures; Fisher syndrome; Friedreich's ataxia; Fibromyalgia; Foville'ssyndrome; Fetal alcohol syndrome; Fragile X syndrome; FragileX-associated tremor/ataxia syndrome (FXTAS); Gaucher's disease;Generalized epilepsy with febrile seizures plus; Gerstmann's syndrome;Giant cell arteritis; Giant cell inclusion disease; Globoid CellLeukodystrophy; Gray matter heterotopia; Guillain-Barré syndrome;Generalized anxiety disorder; HTLV-1 associated myelopathy;Hallervorden-Spatz syndrome; Head injury; Headache; Hemifacial Spasm;Hereditary Spastic Paraplegia; Heredopathia atactica polyneuritiformis;Herpes zoster oticus; Herpes zoster; Hirayama syndrome; Hirschsprung'sdisease; Holmes-Adie syndrome; Holoprosencephaly; Huntington's disease;Hydranencephaly; Hydrocephalus; Hypercortisolism; Hypoxia;Immune-Mediated encephalomyelitis; Inclusion body myositis;Incontinentia pigmenti; Infantile Refsum disease; Infantile spasms;Inflammatory myopathy; Intracranial cyst; Intracranial hypertension;Isodicentric 15; Joubert syndrome; Karak syndrome; Kearns-Sayresyndrome; Kinsbourne syndrome; Kleine-Levin syndrome; Klippel Feilsyndrome; Krabbe disease; Kufor-Rakeb syndrome; Lafora disease;Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; Lateralmedullary (Wallenberg) syndrome; Learning disabilities; Leigh's disease;Lennox-Gastaut syndrome; Lesch-Nyhan syndrome; Leukodystrophy;Leukoencephalopathy with vanishing white matter; Lewy body dementia;Lissencephaly; Locked-in syndrome; Lou Gehrig's disease (See amyotrophiclateral sclerosis); Lumbar disc disease; Lumbar spinal stenosis; Lymedisease—Neurological Sequelae; Machado-Joseph disease (Spinocerebellarataxia type 3); Macrencephaly; Macropsia; Mal de debarquement;Megalencephalic leukoencephalopathy with subcortical cysts;Megalencephaly; Melkersson-Rosenthal syndrome; Menieres disease;Meningitis; Menkes disease; Metachromatic leukodystrophy; Microcephaly;Micropsia; Migraine; Miller Fisher syndrome; Mini-stroke (transientischemic attack); Misophonia; Mitochondrial myopathy; Mobius syndrome;Monomelic amyotrophy; Morvan syndrome; Motor Neuron Disease—seeamyotrophic lateral sclerosis; Motor skills disorder; Moyamoya disease;Mucopolysaccharidoses; Multi-infarct dementia; Multifocal motorneuropathy; Multiple sclerosis; Multiple system atrophy; Musculardystrophy; Myalgic encephalomyelitis; Myasthenia gravis; Myelinoclasticdiffuse sclerosis; Myoclonic Encephalopathy of infants; Myoclonus;Myopathy; Myotubular myopathy; Myotonia congenita; Narcolepsy;Neuro-Behget's disease; Neurofibromatosis; Neuroleptic malignantsyndrome; Neurological manifestations of AIDS; Neurological sequelae oflupus; Neuromyotonia; Neuronal ceroid lipofuscinosis; Neuronal migrationdisorders; Neuropathy; Neurosis; Niemann-Pick disease; Non-24-hoursleep-wake disorder; Nonverbal learning disorder; O'Sullivan-McLeodsyndrome; Occipital Neuralgia; Occult Spinal Dysraphism Sequence;Ohtahara syndrome; Olivopontocerebellar atrophy; Opsoclonus myoclonussyndrome; Optic neuritis; Orthostatic Hypotension; Otosclerosis; Overusesyndrome; Palinopsia; Paresthesia; Parkinson's disease; Paramyotoniacongenita; Paraneoplastic diseases; Paroxysmal attacks; Parry-Rombergsyndrome; PANDAS; Pelizaeus-Merzbacher disease; Periodic paralyses;Peripheral neuropathy; Pervasive developmental disorders; Phantomlimb/Phantom pain; Photic sneeze reflex; Phytanic acid storage disease;Pick's disease; Pinched nerve; Pituitary tumors; PMG; Polyneuropathy;Polio; Polymicrogyria; Polymyositis; Porencephaly; Post-polio syndrome;Postherpetic neuralgia (PHN); Postural hypotension; Prader-Willisyndrome; Primary lateral sclerosis; Prion diseases; Progressivehemifacial atrophy; Progressive multifocal leukoencephalopathy;Progressive supranuclear palsy; Prosopagnosia; Pseudotumor cerebri;Quadrantanopia; Quadriplegia; Rabies; Radiculopathy; Ramsay Huntsyndrome type I; Ramsay Hunt syndrome type II; Ramsay Hunt syndrome typeIII—see Ramsay-Hunt syndrome; Rasmussen encephalitis; Reflexneurovascular dystrophy; Refsum disease; REM sleep behavior disorder;Repetitive stress injury; Restless legs syndrome; Retrovirus-associatedmyelopathy; Rett syndrome; Reye's syndrome; Rhythmic Movement Disorder;Romberg syndrome; Saint Vitus dance; Sandhoff disease; Schilder'sdisease (two distinct conditions); Schizencephaly; Sensory processingdisorder; Septo-optic dysplasia; Shaken baby syndrome; Shingles;Shy-Drager syndrome; Sjögren's syndrome; Sleep apnea; Sleeping sickness;Snatiation; Sotos syndrome; Spasticity; Spina bifida; Spinal cordinjury; Spinal cord tumors; Spinal muscular atrophy; Spinal and bulbarmuscular atrophy; Spinocerebellar ataxia; Split-brain;Steele-Richardson-Olszewski syndrome; Stiff-person syndrome; Stroke;Sturge-Weber syndrome; Stuttering; Subacute sclerosing panencephalitis;Subcortical arteriosclerotic encephalopathy; Superficial siderosis;Sydenham's chorea; Syncope; Synesthesia; Syringomyelia; Tarsal tunnelsyndrome; Tardive dyskinesia; Tardive dysphrenia; Tarlov cyst; Tay-Sachsdisease; Temporal arteritis; Temporal lobe epilepsy; Tetanus; Tetheredspinal cord syndrome; Thomsen disease; Thoracic outlet syndrome; TicDouloureux; Todd's paralysis; Tourette syndrome; Toxic encephalopathy;Transient ischemic attack; Transmissible spongiform encephalopathies;Transverse myelitis; Traumatic brain injury; Tremor; Trichotillomania;Trigeminal neuralgia; Tropical spastic paraparesis; Trypanosomiasis;Tuberous sclerosis; 22q13 deletion syndrome; Unverricht-Lundborgdisease; Vestibular schwannoma (Acoustic neuroma); Von Hippel-Lindaudisease (VHL); Viliuisk Encephalomyelitis (VE); Wallenberg's syndrome;West syndrome; Whiplash; Williams syndrome; Wilson's disease; Y-LinkedHearing Impairment; or Zellweger syndrome.

For example, the methods and systems can be used in subjects having orsuspected of having a neurological disease, disorder, or conditioncomprising a neurodegenerative disease, disorder, or condition. As anexample, a neurodegenerative disease, disorder or condition can beAlzheimer's disease, amyotrophic lateral sclerosis (ALS), Alexanderdisease, Alpers' disease, Alpers-Huttenlocher syndrome,alpha-methylacyl-CoA racemase deficiency, Andermann syndrome, Artssyndrome, ataxia neuropathy spectrum, ataxia (E.g., with oculomotorapraxia, autosomal dominant cerebellar ataxia, deafness, andnarcolepsy), autosomal recessive spastic ataxia of Charlevoix-Saguenay,Batten disease, beta-propeller protein-associated neurodegeneration,Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Corticobasal Degeneration,CLN1 disease, CLN10 disease, CLN2 disease, CLN3 disease, CLN4 disease,CLN6 disease, CLN7 disease, CLN8 disease, cognitive dysfunction,congenital insensitivity to pain with anhidrosis, dementia, familialencephalopathy with neuroserpin inclusion bodies, familial Britishdementia, familial Danish dementia, fatty acid hydroxylase-associatedneurodegeneration, Gerstmann-Straussler-Scheinker Disease,GM2-gangliosidosis (e.g., AB variant), HMSN type 7 (e.g., with retinitispigmentosa), Huntington's disease, infantile neuroaxonal dystrophy,infantile-onset ascending hereditary spastic paralysis, Huntington'sdisease (HD), infantile-onset spinocerebellar ataxia, juvenile primarylateral sclerosis, Kennedy's disease, Kuru, Leigh's Disease,Marinesco-Sjögren syndrome, Mild Cognitive Impairment (MCI),mitochondrial membrane protein-associated neurodegeneration, Motorneuron disease, Monomelic Amyotrophy, Motor neuron diseases (MND),Multiple System Atrophy, Multiple System Atrophy with OrthostaticHypotension (Shy-Drager Syndrome), multiple sclerosis, multiple systematrophy, neurodegeneration in Down's syndrome (NDS), neurodegenerationof aging, Neurodegeneration with brain iron accumulation, neuromyelitisoptica, pantothenate kinase-associated neurodegeneration, OpsoclonusMyoclonus, prion disease, Progressive Multifocal Leukoencephalopathy,Parkinson's disease (PD), PD-related disorders, polycysticlipomembranous osteodysplasia with sclerosing leukoencephalopathy, priondisease, progressive external ophthalmoplegia, riboflavin transporterdeficiency neuronopathy, Sandhoff disease, Spinal muscular atrophy(SMA), Spinocerebellar ataxia (SCA), Striatonigral degeneration,Transmissible Spongiform Encephalopathies (Prion Diseases), orWallerian-like degeneration.

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member can be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group can be included in, or deletedfrom, a group for reasons of convenience or patentability. When any suchinclusion or deletion occurs, the specification is herein deemed tocontain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims.

All publications, patents, patent applications, and other referencescited in this application are incorporated herein by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application or other reference wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes. Citation of a reference herein shallnot be construed as an admission that such is prior art to the presentdisclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing the scope of the present disclosure defined in theappended claims. Furthermore, it should be appreciated that all examplesin the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the present disclosure, and thus can be considered toconstitute examples of modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes can be made in the specific embodiments that are disclosedand still obtain a like or similar result without departing from thespirit and scope of the present disclosure.

Example 1: Focused Ultrasound-Enabled Brain Tumor Liquid Biopsy

The following example describes the detection of a glioblastomabiomarker (mRNA) in an animal model.

Although blood-based liquid biopsies have emerged as a promisingnon-invasive method to detect biomarkers in various cancers, limitedprogress has been made for brain tumors. One major obstacle is theblood-brain barrier (BBB), which hinders efficient passage of tumorbiomarkers into the peripheral circulation. The objective of this studywas to determine whether FUS in combination with microbubbles canenhance the release of biomarkers from the brain tumor to the bloodcirculation. Two glioblastoma tumor models (U87 and GL261), developed byintracranial injection of respective enhanced green fluorescent protein(eGFP)-transduced glioblastoma cells, were treated by FUS in thepresence of systemically injected microbubbles. Effect of FUS on plasmaeGFP mRNA levels was determined using quantitative polymerase chainreaction. eGFP mRNA were only detectable in the FUS-treated U87 mice andundetectable in the untreated U87 mice (maximum cycle number set to 40).This finding was replicated in GL261 mice across three differentacoustic pressures. The circulating levels of eGFP mRNA were 1,500-4,800fold higher in the FUS-treated GL261 mice than that of the untreatedmice for the three acoustic pressures. This study demonstrated thefeasibility of FUS-enabled brain tumor liquid biopsies in two differentmurine glioma models across different acoustic pressures.

Introduction

Mutations in the DNA, changes in epigenomic makeup, and variations ingene expression associated with brain tumors can inform clinicalpractice by providing invaluable information for diagnosis,prognostication, disease monitoring, and development of personalizedtreatment strategies¹. Such molecular biomarkers, which can be examinedin surgical resection or biopsy specimens, are becoming an integralcomponent of clinical practice². However, direct surgical tissue biopsyto determine tumor molecular profiles is associated with potentialcomplications such as hemorrhage and infection³. Furthermore, repeatedtissue biopsies using surgical interventions to assess treatmentresponse and recurrence may not be feasible given the increased risk forcomplications and morbidity, especially for brain tumors. Liquid biopsyoffers a noninvasive approach for detecting circulating molecularbiomarkers.

Although liquid biopsy has been established in clinical care of patientswith various cancer types⁴, limited progress has been made for braintumors². For brain tumor liquid biopsy, the major challenge is thehindrance of tumor biomarker release into the bloodstream by theblood-brain barrier (BBB)⁵. Even though the BBB is partially disruptedin the core part of glioblastoma (the most common type of high-gradeglioma), it remains intact in large parts of glioblastoma and lowergrade diffuse gliomas, which may prevent the efficient passage ofbiomarkers into the blood circulation. In line with this, circulatingtumor DNA (ctDNA) was reported to be detectable in a small fraction ofpatients with advanced gliomas (<10%) as compared to patients with othersolid tumors⁶. It has also been shown that patients with high-gradeglioma have a significantly higher plasma DNA concentration thanpatients with low-grade gliomas. These suggest that the increasedpermeability of BBB associated with the progression of gliomas iscorrelated with the release of biomarkers from the tumor to the blood⁷.It was also found that although D-2-hydroxyglutarate (D2HG) levels havebeen used in the clinic for the diagnosis and monitoring of patientswith IDH1/2-mutant malignancies, D2HG plasma levels in patients withIDH2/2-mutant gliomas are within the normal range, suggesting that theBBB prevents D2HG from entering the blood circulation⁸. Therefore,approaches that can non-invasively enhance the release of biomarkersfrom the brain tumors to the blood circulation would be of significantclinical relevance.

Focused ultrasound (FUS) offers noninvasive and spatially-localizedbiomarker release into the blood stream^(3,9). The initial concept forblood biomarker amplification and localization using ultrasound wasproposed in a study published in 2009, which demonstrated thefeasibility to enhance the release of protein biomarkers from a coloncancer cell line in both ex vivo cell cultures and an in vivo mousetumor model¹⁰. Building on this initial work, in vitro cell culturestudies showed the feasibility of enhancing mRNA biomarker release froma breast cancer cell line using microbubble-enhanced ultrasound¹¹, andthe feasibility of releasing a combination of ovarian cancer biomarkersusing ultrasound¹². Two recent in vivo studies further demonstrated thatultrasound-mediated release of biomarkers into the bloodstream is apromising approach for detecting tumor biomarkers via bloodsampling^(10,13,14). In one of the studies, a chicken embryo tumor modelwas used to show the feasibility of amplifying the release ofextracellular vesicles using high-intensity focused ultrasound (HIFU) incombination with phase-change nanodroplets which changed to microbubblesupon HIFU sonication¹⁴. In the other study, pulsed HIFU with highacoustic pressures was used to induce histotripsy (i.e., a technique formechanical tissue fractionation) in a rat model of prostate cancer, andthis enhanced release of cell-free tumor microRNA into the bloodcirculation¹³. Although promising, these findings cannot be readilyextended to applications in the brain, given challenges inherent to thebrain: first, delivery of acoustic energy to the brain is impeded byattenuation and distortion of acoustic waves by the skull; second,biomarker release from the brain is inherently limited by the presenceof the BBB.

FUS in combination with microbubbles has been studied extensively forinducing BBB opening for noninvasive and localized delivery of drugs inthe blood circulation to the brain parenchyma¹⁵⁻¹⁷. Many studies havebeen performed to optimize the treatment parameters¹⁸⁻²⁰ and evaluatethe short-term and long-term safety profiles²¹⁻²⁴. Ongoing clinicaltrials are evaluating the feasibility and safety of FUS-induced BBBopening in patients with glioblastoma and Alzheimer's disease^(25,26).Although all previous studies focused on exploring the utility ofFUS-induced BBB as a means of targeted delivery of circulatingtherapeutics, here, it was hypothesized that FUS-mediated BBB disruptioncould be viewed as a tool for enhancing “two-way trafficking” betweenbrain and blood. While circulating molecules can be allowed to enter thebrain using FUS-mediated BBB disruption, brain biomarkers (e.g., tumormarkers) can also be released into the blood circulation for liquidbiopsies. Here is disclosed the development of an FUS-enabled braintumor liquid biopsy technique, which uses FUS in combination withmicrobubbles to enhance the release of biomarkers from brain tumors intothe blood circulation for liquid biopsies.

The feasibility of using FUS in combination with microbubbles wasdemonstrated for the local release of mRNA from glioblastoma tumors inmice into the bloodstream for liquid biopsies. Glioblastoma was selectedas the tumor model because it is the most frequent type of primary braincancer in adults and associated with a dismal prognosis²⁷. The biomarkerused was enhanced green fluorescent protein (eGFP) mRNA, which washighly specific to the tumor models used in this study, as the tumormodels were established by the direct injection ofeGFP-luciferase-transduced glioblastoma cells into the mouse brain. Bothhuman glioma U87 cells and murine glioma GL261 cells were used todevelop two mouse models of glioblastoma.

Results

FUS-Enabled Liquid Biopsy in an Orthotopic Human Glioma Xenograft Model

An orthotopic mouse model developed by implantation ofeGFP-luciferase-transduced human glioma cells (U87) into the brains ofnude mice was treated by an ultrasound imaging-guided FUS system (seee.g., FIG. 1A). Local growth of the tumors within the brain was assessedby monitoring luciferase activity using bioluminescent imaging (BLI) andverified using fluorescence imaging of the ex vivo brain slices (seee.g., FIG. 1B). The focus of the FUS transducer (frequency=1.5 MHz) wastargeted at the tumor based on the tumor location identified by the BLI.The acoustic pressure of the FUS pulses was 3.28 MPa and otherparameters were similar to those used for the BBB disruption (duty cycleof 1%, pulse repetition frequency of 1 Hz, and exposure duration of 2min) (see e.g., FIG. 1E)²⁰⁻²⁴. The experimental timeline is shown inFIG. 1F. For the U87 mice, terminal (non-survival) blood collection viacardiac puncture was performed immediately (˜4 minutes) after FUStreatment. Such a short interval was selected in reference to a recentobservation showing shorter collection time after FUS treatment wasassociated with higher RNA yield¹³.

Quantitative polymerase chain reaction (qPCR) was performed to determinerelative circulating levels of eGFP mRNA (target biomarker) normalizedto 5.8S rRNA (internal control) in blood serum. Two pairs of PCR primerswere used for the quantification of eGFP, namely eGFP A and eGFP B(TABLE 1). PCR products for the two eGFP primer pairs were undetectablein the control mice without FUS treatment with the qPCR maximum cyclenumber set to 40 (TABLE 2). Amplification curves of circulating eGFPmRNA in control and treated mice for the two primer pairs are shown inFIG. 2A. Quantification of the eGFP mRNA in blood collected from U87mice found the average±standard deviation of ΔC_(T) for eGFP A and eGFPB were both 30.7±0.8 for the control group without FUS treatment. Forthe FUS-treated mice, the average±standard deviation of ΔC_(T) for eGFPA and eGFP B were 16.6±5.2 and 19.4±5.4, respectively (TABLE 2).

TABLE 1 Forward and reverse primers used in qPCR for eGFP mRNA and 5.8s rRNA. Two primers were used for eGFP quantification, called eGFP A and eGFP B. 5.8s rRNA was used as an  internal control.Primer Forward Reverse eGFP A AGAACGGCATCAAGGTGAA TGCTCAGGTAGTGGTTGC (SEQ ID NO: 1) TCG (SEQ ID NO: 4) eGFP B TATATCATGGCCGACAAGCACTGGGTGCTCAGGTAG A (SEQ ID NO: 2) TGG (SEQ ID NO: 5) 5.8s rRNAGACTCTTAGCGGTGGATCA CGTTCTTCATCGACGCA CT (SEQ ID NO: 3)CGA (SEQ ID NO: 6)

TABLE 2 Summary of normalized cycle threshold, ΔC_(T), for eGFP A andeGFP B in the U87 control mice (C1-C3; n = 3) and treated mice (T1-T6; n= 6). eGFP A eGFP B Mice Identifier ΔC_(T) ΔC_(T) Control Mice C1 29.929.9 C2 30.9 30.9 C3 31.4 31.4 Treated Mice T1 14.8 25.9 T2 19.6 19.3 T325.1 25.1 T4 12.8 13.6 T5 16.4 19.1 T6 10.8 13.3

2^(−ΔC) _(T) was calculated to compare the relative gene expressionlevels of the FUS-treated mice and the control mice (see e.g., FIG. 2Band FIG. 2C). For both eGFP primer pairs, circulating mRNA levels ofeGFP were significantly higher in the FUS-treated group compared withthe untreated control group (eGFP A: p=0.01; eGFP B: p=0.01; one-tailednon-parametric Mann Whitney U Test).

FUS-Enabled Liquid Biopsy in an Orthotopic Murine Xenograft Glioma Model

The second orthotopic glioma model was developed by direct implantationof murine glioma cells (GL261) into the Swiss mice. A magneticresonance-guided FUS (MRgFUS) system was used for the FUS sonication toachieve accurate tumor targeting (see e.g., FIG. 1C). The MRgFUS system,which was operated at 1.44 MHz, was targeted at the center of the tumor.Three groups of GL261 mice were treated by FUS with acoustic pressuresof 1.48 MPa, 2.10 MPa, and 3.34 MPa, respectively. All other parameterswere kept the same as those used in the treatment of U87 mice.Contrast-enhanced MR images were acquired before FUS sonication toidentify the location of the tumor for FUS targeting and after FUSsonication to verify accurate tumor targeting by the FUS (see e.g., FIG.1D).

Quantification of the eGFP mRNA in the blood collected from GL261 micefound the average±standard deviation of ΔC_(T) for eGFP A and eGFP Bwere both 23.6±0.2 for the control group. The mean±standard deviation ofΔC_(T) for eGFP A was 11.6±0.1 for the 1.48 MPa group, 12.5±0.4 for the2.01 MPa group, and 12.2±0.1 for the 3.34 MPa group (TABLE 3). Themean±standard deviation of ΔC_(T) for eGFP B were 12.2±0.3, 13.7±0.7,and 13.0±0.2 for the three groups, respectively (TABLE 3).

TABLE 3 Summary of normalized cycle threshold, ΔC_(T), for eGFP A andeGFP B in the GL261 control mice (C1-C3; n = 3) and treated mice withdifferent acoustic pressures. eGFP A eGFP B Mice Identifier ΔC_(T)ΔC_(T) Control Mice C1 23.6 23.6 C2 23.9 23.8 C3 23.4 23.4 Treated Mice1.48 MPa 11.6 12.4 1.48 MPa 11.5 11.8 1.48 MPa 11.7 12.3 2.10 MPa 12.513.9 2.10 MPa 12.0 12.8 2.10 MPa 12.9 14.5 3.34 MPa 12.3 13.2 3.34 MPa12.2 13.1 3.34 MPa 12.1 12.8

Regardless of acoustic pressure, circulating eGFP levels weresignificantly higher (1,500-4,800 fold higher) in the FUS-treated groups(n=9 in total, eGFP A: p=0.0045; eGFP B: p=0.0045; one-tailed MannWhitney U Test) relative to the control group (n=3, FIG. 3). Theexpression levels of mice treated at the lowest pressure (n=3; 1.48 MPa)were significantly higher than those of the other two groups for eGFP Aand eGFP B (n=6; eGFP A: 1.7 fold increase in average, p=0.012; eGFP B:2.2 fold increase in average; p=0.012; one-tailed Mann Whitney U Test).This finding suggests that the relatively lower pressure (1.48 MPa) wasmore efficient in releasing eGFP mRNA from the tumor than the relativelyhigher pressures (2.10 MPa and 3.34 MPa). Additionally, these MRI datafound that the MR contrast enhancement ratios (calculated by theintensities of the MR images acquired after FUS treatment divided by theintensities of the images obtained before FUS treatment) were notsignificantly different among the three pressure groups.

Histological Examination

H&E staining of the GL261 mouse brains found red blood cellextravasation in all mice treated with FUS (see e.g., FIG. 4). Moresevere hemorrhage was observed in brain slices obtained from micetreated with the relatively higher pressures (2.10 MPa and 3.34 MPa)than mice treated with the relatively lower pressure (1.48 MPa).Vascular damage was expected as the acoustic pressures used in thisstudy were higher than the pressures normally used for the BBB openingwithout causing vascular damage. Of note, hemorrhage was not observed inthe U87 mice treated by FUS at 3.28 MPa. The short interval between FUSsonication and animal scarification in the U87 mice (about 4 minutes vs.20 minutes in GL261 mice) may have precluded the appearance of red bloodcells in the brain slices even in the presence of tissue damage.

Discussion

This study demonstrated the use of FUS-enabled brain liquid biopsy usingtwo glioma mouse models. FUS in combination with microbubbles achievednoninvasive and spatially-localized biomarker release from the braintumor into the bloodstream. The noninvasive nature of the disclosed FUStechnique is especially advantageous over conventional neurosurgicaltissue biopsies. Moreover, the technique presents a unique advantage inthe assessment of spatially heterogeneous tumors. Tumor heterogeneity,which is a hallmark of glioblastoma²⁸, poses a significant challenge tocancer biomarker research²⁹. FUS can precisely target differentlocations of the tumor, thereby causing biomarkers to be released in aspatially-localized manner. By targeting multiple tumor regions in asingle FUS session, the technique can be used to capture and analyzespatially heterogeneous biomarkers in a single liquid biopsy sample.Another potential functionality would be to perform multiple FUSsessions, each followed by a liquid biopsy, in order to detect specificbiomarkers for each spatial location of the tumor to better understandthe spatial heterogeneity of the tumor.

Both ultrasound-imaging guided FUS and MRgFUS treatment yieldedsubstantial increase in eGFP RNA levels. However, the standarddeviations of the qPCR measurement results for FUS-treated GL261 micewere lower than those of the FUS-treated U87 mice (see e.g., FIG. 2,FIG. 3). This decrease in variations of the experimental results wasassociated with the use of the MRgFUS system, which improved the tumortargeting accuracy compared with the ultrasound imaging-guided FUSsystem.

Although damage was observed, the FUS-enabled brain liquid biopsytechnique has clear safety benefits compared over craniotomy forsurgical biopsy of brain tissue. Moreover, the finding that among thethree acoustic pressures the lowest pressure (1.48 MPa) achieved thehighest eGFP mRNA expression level is important. It suggests thatvascular damage associated with FUS treatment at the relatively higheracoustic pressures (2.10 MPa and 3.34 MPa) may hinder the efficientpassage of tumor biomarkers into the peripheral circulation. It alsosuggests that successful biomarker release may be achievable withacoustic pressures lower than 1.48 MPa, which can improve the safety ofthe FUS technique. Relatively higher acoustic pressures (1.45-3.34 MPa)were used in the current study than what is commonly used for the BBBdisruption (e.g., 0.45 MPa), because in this study it was intended toenhance the interaction between microbubbles and the brain tissues.Optimization of the acoustic parameters (e.g., acoustic pressure, pulserepetition frequency, pulse length, and sonication duration) andmicrobubble parameters (e.g., size and dose) can be performed to explorethe potential to achieve enhanced biomarker release with minimal or notissue damage.

The disclosed technique can be optimized by assessing the efficiency ofbiomarker release under different FUS and microbubble parameters (seee.g., Example 2). Specifically, the release of biomarkers using FUS withlower pressures (e.g., 0.45 MPa) that are commonly used for BBBdisruption without causing vascular damage can be evaluated. Second, theshort-term and long-term safety of the FUS brain liquid biopsy techniquecan be assessed. Extracranial metastases of glioblastoma are rare³¹, andrisk for tumor spread after surgical brain biopsies is considerednegligible. It is expected that the FUS liquid biopsies will not inducemetastatic spread. Nevertheless, studies can be performed to determinethe potential of metastasis associated with the FUS treatment. Third,the terminal cardiac puncture was used for blood collection as mice havea small total blood volume (˜1.5 mL). Repeated blood sample collectionwas not feasible using the mouse model, but could be accomplished inhumans and larger animals. Larger animal models (e.g., rats, pigs) canbe used to collect blood samples at multiple time points after the FUStreatment. These blood samples can be used to assess the temporaldependency of the amount of biomarkers released by the FUS treatment andestablish the optimal blood collection time. The larger animal modelscan be used to evaluate the repeatability of this technique byperforming the FUS treatment on the same animal on multiple days.Fourth, the contrast-enhanced MR images acquired before and after theFUS treatment were quantified (see e.g., FIG. 1D). There was nosignificant difference in the MR contrast enhancement ratios among thethree pressure groups (1.48 MPa, 2.10 MPa, and 3.34 MPa). This findingwas not consistent with the quantification results of eGFR mRNAexpression level shown in FIG. 3, which found the eGFR mRNA expressionlevel in the 1.48 MPa group was significantly higher than that of theother two higher pressure groups. Future studies will determine whenlower acoustic pressures are used whether contrast-enhanced MRI is auseful tool in predicting the amount of biomarkers released by FUS.Fifth, the molecular marker (eGFP) was used to demonstrate thefeasibility of FUS in noninvasive and spatially-focused liquid biopsy.Studies can use the same methods to assess the other tumor markers(e.g., DNA-based markers). The exact mechanism by which FUS-enabledrelease of molecular biomarkers is unknown. It is presently believedthat the mechanism may be that the FUS-induced BBB disruption opens a“two-way door,” allowing “two-way trafficking” between the brain andblood. Future studies will explore the potential mechanisms ofFUS-enabled biomarker release.

Conclusions

It was demonstrated that the combination of FUS and microbubbles allowsdetection of tumor-specific eGFP mRNA in the bloodstream that isotherwise undetectable. The presently disclosed findings establishedthat FUS-mediated BBB disruption could enhance brain-to-bloodtrafficking. FUS may offer an enabling technique for noninvasive andregionally-specific brain tumor liquid biopsy that can be utilized inpersonalized brain cancer patient care.

Methods

Orthotopic Mouse Glioblastoma Models

All animal procedures were reviewed and approved by the InstitutionalAnimal Care and Use Committee of Washington University in St. Louis inaccordance with the National Institutes of Health Guidelines for animalresearch.

Two orthotopic mouse glioblastoma models were developed: (i) NCI athymicNCr-nu/nu mice (Strain 553, Charles River Laboratory, Wilmington, Mass.,USA) injected with U87 human glioblastoma cells; and (ii) NIH Swiss mice(Strain 550, Charles River Laboratory, Wilmington, Mass., USA) implantedwith GL261 murine glioblastoma cells. Mice were anesthetized and fixedinto a stereotactic head frame. A paramedian incision was made on thescalp, and a 1-mm burr hole was drilled 2 mm posterior and 1.5 mmlateral to the bregma. eGFP-Luciferase-transduced glioblastoma cells(U87 or GL261) were mixed with Corning™ Matrigel (Catalog 356231,Corning Life Science, New York, USA) and injected through the burr holeusing a syringe. The burr hole was sealed with bone wax, and the skinincision was glued together with tissue glue. The growth of the tumorwas monitored using IVIS® Spectrum In Vivo Imaging System (Model 124262,PerkinElmer, Ohio, USA) once every week for four weeks. At around fifthweek after tumor cell implantation, mice were recruited in the studydescribed below.

Ultrasound Imaging-Guided FUS Treatment of U87 Mice

A total of nine mice with orthotopic U87 glioblastoma tumors weredivided into two groups: treatment group (n=6) and control group (n=3).The treatment group was treated with FUS spatially targeted at the tumorsite after intravenous injection of microbubbles. The control groupreceived no FUS. For FUS treatment, mice were first anesthetized with 2%isoflurane and placed on a stereotactic frame. A FUS system (VIFU 2000;Alpinion US Inc., Bothell, Wash., USA) sonicated the tumor using thefollowing parameters: frequency=1.5 MHz, peak negative pressure=3.28MPa, pulse length=10 ms, pulse repetition frequency=1 HZ, duration=30 sat each location, and 4 separate locations were treated for each tumor(see e.g., FIG. 1A and FIG. 1E). The pressure amplitudes and beamdimensions of the FUS transducer were calibrated using a needlehydrophone (Onda, CA, USA) in a degassed water tank before theexperiment. The pressures reported here were the peak negative pressuresmeasured in water. The full width at half maximum (FWHM) of the axialbeam and lateral beam were 6.04 mm and 0.62 mm, respectively. Before FUSsonication, microbubbles manufactured in house³² were injected into themouse tail vein at a concentration of 8×10⁸ microbubbles per mL and avolume of 30 μL per mouse. The in-house manufactured microbubblescomprised of a 90 mol % 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC) and 10 mol %1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylenegly-col)2000] (DSPE-PEG2000) (Avanti Polar Lipids, Alabaster, Ala., USA)lipid-shell and a perfluorobutane (FluoroMed, Round Rock, Tex., USA)gas-core. These microbubbles had a median diameter of 4-5 mm, which wereisolated from a poly-dispersed microbubble distribution using adifferential centrifugation method³³.

MRgFUS Treatment of GL261 Mice

A total of 12 mice with implantation of GL261 glioblastoma tumors in thebrain were split into four groups: control group (n=3) and threetreatment groups (n=3 for each group). The three treatment groups weretreated using a clinical MRgFUS system (Sonalleve V2, Profound MedicalInc., Mississauga, Canada) equipped with a dedicated small animaladapter (FUS Instruments Inc., Toronto, Ontario, Canada) (see e.g., FIG.1C)³⁴. The Sonalleve MRgFUS system included a 256-element phased arraytransducer mounted to a five-axis robot positioner and located inside amodified MRI patient table. The acoustic fields generated by the phasedarray transducer were calibrated by a fiber optic hydrophone using thepreviously published method³⁵. The FWHM of the axial beam and lateralbeam were 12.10 mm and 1.37 mm, respectively. The MRgFUS system wasintegrated into a clinical MRI scanner (Ingenia 1.5T, Philips, Best, theNetherlands). The small animal adapter included a frame to hold ananimal, a water reservoir, and a small animal MRI coil (imaging probe).

For the treatment of GL261 mice, mice were anesthetized with 1-2%isoflurane and placed on the small animal adapter. Optimark(gadoversetamide, a gadolinium-based contrast agent, 0.5 mmol/ml) wasinjected intravenously into the mice through the tail vein at a dose of0.1 mmol/kg. Contrast-enhanced 3D, T₁-weighted MRI images of the mousebrain were acquired for treatment planning, and the FUS targetedlocation was selected to be the center of the tumor (see e.g., FIG. 1D).After intravenous injection of the same dose of microbubbles as that wasused for U87 mice, the GL261 mice were treated by the MRgFUS systemusing the following parameters: frequency=1.44 MHz, pulse length=10 ms,pulse repetition frequency=1 Hz, and duration=2 min. The three treatmentgroups were treated at different peak negative pressures: 1.48 MPa, 2.10MPa, and 3.34 MPa, respectively. After treatment, contrast-enhanced MRIimages were acquired for confirming successful tumor targeting by theFUS (see e.g., FIG. 1D). Blood samples of 500-800 μL were collected fromthe heart about 20 min after the FUS treatment and prepared for qPCRanalysis of eGFP mRNA.

Analysis of eGFP mRNA

Blood samples of 500-800 μL were collected from the heart about 4 min(U87) or 20 min (GL261) after the FUS treatment and prepared for qPCRanalysis of eGFP mRNA. All the collected blood was centrifuged at 3,000rpm for 10 minutes. The supernatant was collected. RNA samples werepurified using the miRNeasy serum/plasma kit (Catalog no. 217184,Qiagen, USA). Agencourt RNAClean XP beads (Catalog no. A63987, BeckmanCoulter Inc., USA) were used to further purify the RNA. The RNA was thenreverse transcribed to cDNA using the Applied Biosystems™ high-capacitycDNA reverse transcription kit (Catalog no. 4368814, Thermo FisherScientific, USA). Two eGFP primer pairs (TABLE 1) were designed usingOligoPerfect™ Designer (ThermoFisher Scientific, USA). 5.8s rRNA wasused as an internal amplification control with its forward and reverseprimer sequences also listed in TABLE 1. The quantitative real-time PCRwas performed using SYBR™ Green PCR master mix (Applied Biosystems™).All of the PCRs were performed on a 7900HT Fast Real-Time PCR System(Catalog #4329001, Thermo Fisher Scientific, USA) using the followingprotocol: the reaction mixture was heated at 95° C. for 10 min, followedby 40 cycles of 95° C. for 5 s and 60° C. for 30 s. Amplification anddissociation curves generated by the SDS2.3 (Applied Biosystems)software were used for gene expression analysis.

Duplicate reactions were run for each sample and each primer set. 5.8SrRNA was used as the internal control to normalize the PCR data bycalculating cycle threshold change (ΔC_(T)) by subtracting C_(T) of theeGFP (C_(T,eGFP)) by the C_(T) of housekeeping gene, 5.8s rRNA(C_(T,5.8S)). The gene expression level was determined using the2^(−ΔCT) method: 2^(−ΔC) ^(T) =2^(−(C) ^(T-eGFP) ^(−C) ^(T,5.8s) ⁾.Maximum cycle number was set to 40. In order to assess the reliabilityof 5.8S rRNA as the internal control, 2^(−C) _(T) was calculated andcompared for the treated and control groups³⁶. The 2^(−C) _(T) of thecontrol mice (1.8×10⁻³±8.6×10⁻⁴) was not significantly different fromthe 2^(−C) _(T)Of the FUS-treated mice (1.1×10⁻³±1.8×10⁻³; p=0.38;two-tailed Mann-Whitney U test).

Histological Analysis

After blood collection, all the mice were transcardially perfused with0.01 M phosphate-buffered saline and then with 4% paraformaldehyde, andtheir brains were harvested and prepared for paraffin sectioning. Themouse brains were horizontally sectioned to 15 μm slices and used forH&E staining.

Statistical Analysis

GraphPad Prism (v6.04, La Jolla, Calif., USA) and R (v3.4.1, R CoreDevelopment Team, 2017) were used to perform statistical analyses. Giventhe non-normality of 2^(−ΔCT) distribution (Shapiro-Wilk test <0.05),group comparison was made using a non-parametric Mann Whitney U Test. Ap-value <0.05 was used to determine statistical significance.

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Multi-modality safety assessment of blood-brain barrier opening    using focused ultrasound and definity microbubbles: a short-term    study. Ultrasound Med. Biol. 36, 1445-59 (2010).-   23. Downs, M. E. et al. Long-term safety of repeated blood-brain    barrier opening via focused ultrasound with microbubbles in    non-human primates performing a cognitive task. PLoS One 10,    e0125911 (2015).-   24. Olumolade, O. O., Wang, S., Samiotaki, G. & Konofagou, E. E.    Longitudinal motor and behavioral assessment of blood-brain barrier    opening with transcranial focused ultrasound. Ultrasound Med. Biol.    42, 1-13 (2016).-   25. Radovini, N. N. World first: blood-brain barrier opened    non-invasively to deliver chemotherapy—Sunnybrook Hospital. (2015)    Available at https://sunnybrook.ca/media/item.asp?i=1351. (Accessed:    4 Mar. 2018)-   26. Nadia Norcia Radovin. First Alzheimer's patient treated with    focused ultrasound to open the blood-brain barrier. (2017).    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Example 2: Optimization of Focused Ultrasound-Enabled Brain Tumor LiquidBiopsy (FUS-Lbx)

The following example describes the optimization of the FUS-LBxtechnique by investigating the effects of FUS acoustic pressure on thetumor biomarker release level and potentially associated hemorrhageburden.

The development of noninvasive approaches for brain tumor diagnosis andmonitoring continues to be a major medical challenge. Althoughblood-based liquid biopsy has received considerable attention in variouscancers, limited progress has been made for brain tumors, at leastpartly due to the hindrance of tumor biomarker release into theperipheral circulation by the blood-brain barrier. As shown in Example1, the use of focused ultrasound-enabled brain tumor liquid biopsy(FUS-LBx) was demonstrated. The objective of this example was tooptimize the FUS-LBx technique by investigating the effects of FUSacoustic pressure on the tumor biomarker release level and potentiallyassociated hemorrhage burden. Mice with orthotopic implantation ofenhanced green fluorescent protein (eGFP)-transfected murine gliomacells were treated using magnetic resonance (MR)-guided FUS system inthe presence of systemically-injected microbubbles at three peaknegative pressure levels (0.59, 1.29, and 1.58 MPa). Plasma eGFP mRNAlevels were quantified with the quantitative polymerase chain reaction(qPCR). Contrast-enhanced MR images were acquired before and after theFUS treatment. FUS at 0.59 MPa resulted in increased plasma eGFP mRNAlevels, comparable to those of higher acoustic pressures (1.29 MPa and1.58 MPa). Hemorrhage associated with FUS at 0.59 MPa was significantlylower than that with higher acoustic pressures and not significantlydifferent from the control group. MRI analysis revealed thatpost-sonication intratumoral and peritumoral hyperenhancement wereassociated with the level of FUS-induced biomarker release and theextent of hemorrhage. Taken together, this study suggests that FUS-LBxtechnique can be optimized to be a safe and effective image-guidedbiomarker release technique.

Introduction

Central nervous system (CNS) tumors are significant causes of cancermorbidity and mortality, especially in children and young adults wherethey account for ˜20-30% of cancer deaths¹. Noninvasive neuroimagingmodalities (e.g., magnetic resonance imaging (MRI), and computerizedtomography (CT)) are used to evaluate tumor lesions, but observedchanges, especially after treatment, can be difficult to interpret².Surgical resection or stereotactic biopsy is typically performed forhistologic confirmation and increasingly for genetic profiling. However,tissue biopsy requires brain surgery and can be associated with adverseeffects such as hemorrhage and infection³. Repeated tumor biopsies thatmay be needed for tracking tumor evolution, treatment response, andtumor recurrence are often not feasible. Furthermore, tissue biopsiesmay be challenging when tumors are located at difficult locations in thebrain or CNS or patients that are too ill to tolerate invasiveprocedures⁴.

Noninvasive blood-based liquid biopsies are a rapidly emerging strategyto provide genetic tumor profiling that can be used for treatmentselection, treatment monitoring, residual disease detection, andasymptomatic individual screening⁵. Some success has been achieved inintegrating blood-based liquid biopsy into the routine clinicaldiagnostics^(6,7). However, limited progress has been made for braintumors. Although several groups have reported biomarker detection in thecerebrospinal fluid (CSF) of patients with brain tumors^(8,9), obtainingCSF via lumbar puncture remains an invasive procedure and is oftenunsafe in these settings. Blood-based liquid biopsies are noninvasive,but there remain multiple hurdles for their application in brain tumors.First, the blood-brain barrier (BBB) restricts the release of largemolecules such as DNA and RNA from the tumor to the peripheralcirculation¹⁰. Even though partial BBB disruption is a core feature ofglioblastoma (the most common type of high-grade glioma) at the latestage of the tumors, the BBB remains intact in the early stage and thelow-grade diffuse gliomas¹¹. Second, brain tumors, especiallyglioblastomas, are heterogeneous with spatially distinct molecularprofiling¹². Localization of tumor areas that harbor specific mutationsis not possible using conventional methods of liquid biopsy, as thesemethods are inherently spatially agnostic. Third, many tumor markers,such as some cell-free RNAs¹³ and DNAs¹⁴, have short half-lives inblood. Detection of these biomarkers can be enhanced by stimulatingtheir release from the tumor to the circulation and preciselycontrolling the blood-collection time to be shorter than their lifetimesin the blood. Therefore, a noninvasive, spatially resolved, andtemporally controlled liquid biopsy technique is in great need toimprove the clinical care of patients with CNS tumors.

Focused ultrasound-enabled brain tumor liquid biopsy (FUS-LBx) (seee.g., Example 1) offers a technique that can provide noninvasive,spatially resolved, and temporally controlled brain tumor liquidbiopsies. FUS has emerged as a technology with the potential tononinvasively exert mechanical and thermal effects on the brain tissue.When coupled with intravenously injected microbubbles, low-intensitypulsed FUS-induced mechanical effects can transiently and non-invasivelyopen the BBB without causing any vascular or tissue damage¹⁶⁻¹⁸. InExample 1, it was demonstrated that FUS in combination with microbubblescould also be exploited to release biomarkers from brain tumors inmurine glioblastoma models¹⁵. This approach is different from previouslyreported FUS-facilitated liquid biopsy techniques developed for in vivobiomarker release from cancers outside the brain¹⁹⁻²¹. In one of thosereports, pulsed high-intensity focused ultrasound (HIFU) with highacoustic pressures (ultrasound frequency=1.5 MHz, peak compressionalfocal pressure=90 MPa, and peak rarefactional focal pressure=17 MPa) wasused to induce histotripsy (i.e., a technique for mechanical tissuefractionation) in a rat model of prostate cancer, and this enhanced therelease of cell-free tumor microRNA into the blood circulation¹⁹. Inanother study, a chicken embryo tumor model was used to show thefeasibility of amplifying the release of extracellular vesicles usingHIFU (ultrasound frequency=1.15 MHz and peak to peak pressure was withinthe range of 10-30 MPa) in combination with phase-change nanodropletswhich changed to microbubbles upon HIFU sonication²⁰. In a recent study,two protein biomarkers were found to be significantly increased in theplasma of patients undergoing HIFU thermal ablation (ultrasoundfrequency=1.1 MHz and power of 100-200 W) of uterine fibroids²¹. Allthese previous studies used HIFU to induce permanent mechanical orthermal disruption of the tumor to enhance the release of tumorbiomarkers. The tissue damaging effect limits the application of thesetechniques as diagnostic tools in a sensitive organ such as the brain.In contrast, the FUS-LBx technique described here uses low-intensitypulsed FUS, which has the potential advantage of enabling the biomarkerrelease without causing tissue damage. However, in Example 1, theacoustic pressures used were intentionally selected to be relativelyhigh (1.52-3.53 MPa) to increase the chance of success in releasingbiomarkers. As expected, hemorrhage was found in the FUS-targeted brainarea.

The objective of this example was to investigate the effects of FUSacoustic pressure on the level of tumor biomarker release and the extentof associated hemorrhage in order to optimize the FUS-LBx technique. Itwas sought to determine the optimal ultrasonic pressure for FUS-LBx thatcan sufficiently increase plasma levels of the tumor biomarkers while atthe same time minimize the risk of hemorrhage in the brain. It wasfurther explored whether post-sonication changes in tumor MR contrastenhancement can predict successful biomarker release for the futuredevelopment of image-guided FUS-LBx technique.

Results

Effect of the Peak Negative Pressure on FUS-LBx Yield

Mice with orthotopic implantation of enhanced green fluorescent protein(eGFP)-transfected murine glioblastoma cells were recruited into thisstudy when the maximum diameter of the tumor reached 2 mm. The mice weretreated by an MR-guided FUS system (see e.g., FIG. 5A, FIG. 5B) at threedifferent peak negative acoustic pressure (PNP) levels: (i) 0.59 MPa;(ii) 1.29 MPa; (iii) 1.58 MPa (n=5 in each group). The acoustic pressureoutput of the FUS transducer was calibrated using a fiber-optichydrophone following a previously published method (see e.g., FIG.5C)²².

Two primer sets were used to improve the robustness of the quantitativepolymerase chain reaction (qPCR) results. When compared to the controlgroup (no FUS or microbubble), all three FUS-treated groups demonstratedsignificant increases in plasma eGFP mRNA levels (all FUS-treated micevs. control group: one-tailed p=6.5×10⁻⁵ for both primer sets: primers Aand B; TABLE 4, FIG. 6). When compared with the control group, therewere 55-fold (Primer A: one-tailed p=0.004) and 221-fold (Primer B:one-tailed p=0.004) increase in plasma eGFP mRNA levels in mice thatreceived FUS with the lowest PNP (0.59 MPa). Plasma eGFP mRNA levels inmice treated with 1.29 MPa and 1.58 MPa achieved respectively about2,000-fold and 8,000-fold average enhancement relative to the controlgroup, respectively (TABLE 4). For primer A, plasma eGFP mRNA levelswere significantly greater with high acoustic pressures when compared to0.59 MPa-FUS treated mice (1.29 MPa vs. 0.59 MPa: 35-fold higher,one-tailed p=0.004; 1.58 MPa vs. 0.59 MPa: 151-folder higher, one-tailedp=0.004). These effects were less pronounced when eGFP mRNA levels weremeasured using primer B (1.29 MPa vs. 0.59 MPa: 5-fold higher,one-tailed p=0.048; 1.58 MPa vs. 0.59 MPa: 22-fold higher, one-tailedp=0.11).

TABLE 4 Group average ± standard deviation of eGFP RNA levels,hemorrhage densities, and tumor MRI enhancement. Group Control 0.59 MPa1.29 MPa 1.58 MPa eGFP mRNA 2.2 × 10⁻⁸ ± 1.2 × 10⁻⁶ ± 4.2 × 10⁻⁵ ± 1.8 ×10⁻⁴ ± level 3.1 × 10⁻⁸ 9.1 × 10⁻⁷ 4.7 × 10⁻⁵ 9.1 × 10⁻⁷ (Primer A),2^(−ΔCT) eGFP mRNA 2.2 × 10⁻⁸ ± 4.8 × 10⁻⁶ ± 2.5 × 10⁻⁵ ± 1.0 × 10⁻⁴ ±level 1.5 × 10⁻⁸ 5.5 × 10⁻⁶ 2.7 × 10⁻⁵ 1.7 × 10⁻⁴ (Primer B), 2^(−ΔCT)Hemorrhage 0.12 ± 0.06 0.17 ± 0.08 0.95 ± 0.6  1.7 ± 1.0 density (%)Tumor MR 12.5 ± 4.1  13.7 ± 3.9  13.5 ± 3.6 13.7 ± 2.2 enhancement

Effect of the Peak Negative Pressure on Brain Hemorrhage

Hematoxylin and eosin (H&E) stained sections of the brain were examinedfor the presence of microhemorrhages. Representative images of theH&E-stained sections are shown in FIG. 7A-FIG. 7D. After colordeconvolution, areas of microhemorrhage were identified using thepositive-pixel count algorithm (see e.g., FIG. 7E). Microhemorrhagedensity was calculated as the proportion of surface area ofmicrohemorrhage to the total surface area of the evaluated brain tissuein a given slice. There was no significant difference in microhemorrhagedensity between mice treated with 0.59 MPa FUS and the control mice(TABLE 4, FIG. 7F). The microhemorrhage density was significantly higherin 1.29 MPa and 1.58 MPa groups than the 0.59 MPa and control groups.Microhemorrhages seen in the control mice were predominantly scatteredin the tumor. In addition to scattered intratumoral microhemorrhage,peritumoral hemorrhage near the interface of the tumor and normal brainparenchyma was seen in 1 out of 5 mice treated with 0.59 MPa FUS as wellas in 4 out of 5 mice in the 1.29 MPa and 1.58 MPa FUS-treated groups.Large variations in the hemorrhage density were observed within eachgroup, especially at higher pressures (1.29 MPa and 1.58 MPa).

Predicting FUS-LBx Yield Using MRI

Increased MRI image intensity on contrast-enhanced MR images was seenboth within the tumor and in the peritumoral area of the tumor aftersonication (see e.g., FIG. 8A). Because there is a significantdifference between intratumoral and peritumoral enhancement beforesonication, it was opted to evaluate post-sonication intratumoral andperitumoral enhancement separately. Possible associations betweenpost-sonication enhancements and microhemorrhage density and FUS-LBxyield were interrogated. Greater sonication-induced intratumoral andperitumoral contrast enhancement were associated with higher hemorrhageburden (see e.g., FIG. 8B, FIG. 8C) and greater post-sonication plasmaeGFP mRNA levels detected using primer A (see e.g., FIG. 8D, FIG. 8E).The correlation coefficients were lower for the eGFP levels (r=0.52 forintratumoral contrast enhancement and r=0.64 for peritumoralenhancement) than the hemorrhage burden (r=0.71 for intratumoralcontrast enhancement and r=0.74 for peritumoral enhancement). Similarassociations were seen between intratumoral/peritumoral enhancements andeGFP mRNA levels measured with primer B (r=0.51 for intratumoralcontrast enhancement and r=0.56 for peritumoral enhancement). Thecorrelation coefficients for all the above analysis were slightly higherwith peritumoral contrast enhancement than intratumoral contrastenhancement.

Discussion

In a murine orthotopic glioblastoma model, it was demonstrated thatFUS-LBx technique could be applied with low acoustic pressure withoutsignificant sonication-induced hemorrhage. The findings from the priorstudy¹⁵ (Example 1) were replicated and expanded and used PNPs(0.59-1.58 MPa) that were lower than those in the previous study(1.52-3.53 MPa). Within 0.59-1.58 MPa, higher pressures were associatedwith a trend of higher biomarker release level but also increased riskof brain hemorrhage.

The results of this study demonstrated that the FUS-induced release ofeGFP mRNA from the tumor into the blood circulation is higher when FUSwith high PNPs (1.29 and 1.58 MPa) is applied compared with relativelylow pressure (0.59 MPa) (see e.g., FIG. 6). The reason for thisdifference might be that higher PNPs may lead to an increased likelihoodof microbubbles undergoing violent inertial cavitation that can, inturn, induce greater disruption of the BBB²³⁻²⁶. In this way, there is ahigher possibility that a higher amount of eGFP mRNA can be released tothe bloodstream. In Example 1, the eGFP mRNA level was found to not bepressure dependent within the range of 1.52-3.53 MPa. Taken together,these findings suggest that as FUS pressure increases the biomarkerrelease level increases and then reaches a plateau. This may be in partdue to potential paradoxical effects of increasing PNP on FUS-LBx yield.On the one hand, higher FUS PNP can result in greater BBB disruption,while on the other hand, it can result in vascular damage and decreasedtumor perfusion, and hinder tumor biomarker release into peripheralcirculation. Moreover, the relative expression levels of eGFP(normalized to 5.8S rRNA) were used in the plasma to index FUS-LBxdiagnostic yield. It is possible that at higher PNPs, potentiallyincreased levels of eGFP are offset by possible increases in 5.8S rRNAreleased from damaged brain tissue.

For the evaluation of the brain hemorrhage, the results showed asignificantly higher microhemorrhage when comparing the 1.29 MPa and1.58 MPa group with the 0.59 MPa group or the control group (see e.g.,FIG. 7). Although there was no statistically significant difference inthe extent of microhemorrhage between the control group and theFUS-treated group receiving 0.59 MPa PNP, microhemorrhage beyond thebounds of the tumor was observed along the interface between the tumorand normal brain tissue in one out of five cases. This pattern ofhemorrhage was more pronounced with higher FUS PNPs (see e.g., FIG. 7Cand FIG. 7D). This pattern of hemorrhage in the tumor periphery mayresult from the immaturity and instability of peritumoral vasculaturerendering them more susceptible to mechanical injury induced by FUS.

Relatively large variations within each group were observed in theanalysis of both FUS-LBx yield and FUS-induced hemorrhage. Suchvariations among repeated FUS treatments have been reported before inFUS-induced BBB disruption experiments. It can be caused by variationsin parameters that are hard to control, such as the size distribution ofmicrobubbles that reach the targeted brain location²⁷, circulatingmicrobubble concentration in blood²⁸, blood vessel density within thetreated region of the tumor²⁹, and heterogeneous acoustic property ofskull³⁰. Therefore treatment monitoring techniques, such as passivecavitation imaging³¹⁻³³, can be used to detect and characterizepotential variations among repeated FUS treatment. Feedback controlalgorithms can also be implemented based on cavitation monitoring tocontrol the FUS output during the treatment to minimize variationsassociated with the FUS treatment^(34,35).

This study also demonstrated that greater contrast enhancement wasassociated with both a greater level of eGFP plasma level and a higherhemorrhage density (see e.g., FIG. 8). Post-sonication intratumoral andperitumoral enhancements were both associated with higher released tumormarkers in plasma. At the same time, post-sonication enhancement withinthe tumor and in its periphery were associated with microhemorrhagedensity. MR imaging markers that can predict safe and successful liquidbiopsy are essential for translation of FUS-LBx to the clinic, as theycan inform the clinician for sufficiency of FUS application for liquidbiopsy or need for a higher magnitude of PNP. Future studies will beperformed to explore the potential of MRI in the assessment of FUS-LBxoutcome and safety. MRI can be combined with passive cavitation imagingto develop image-guided FUS-LBx which employs real-time passivecavitation imaging for FUS-LBx treatment monitoring and feedback controland utilizes MRI for treatment outcome and safety assessment.

There are limitations to be considered. First, plasma mRNA levels ofeGFP were used that were uniquely expressed in the tumor cells toevaluate FUS-mediated liquid biopsy. While this approach is adequate todetermine the effectiveness of FUS-LBx, it may not fully recapitulatethe clinical settings for liquid biopsy. However, the efficacy ofFUS-LBx can be interrogated to discern disease-causing variations ofnaturally occurring tumor-specific biomarkers³⁶. Of note, biomarkers canbe evaluated within a short interval from their release to thecirculation after FUS application. This permits the detection ofshort-lived molecules such as mRNAs and some proteins. Second, similarto Example 1, blood was collected using terminal cardiac puncture asmice have a small total blood volume (˜1.5 mL). This approach precludesrepeated blood sampling to monitor plasma mRNA levels over timefollowing sonication or to determine the effects of serial FUS-mediatedBBB opening on plasma tumor biomarkers. Therefore, in order to determinethe temporal pattern of released tumor biomarkers and feasibility ofserial liquid biopsy, FUS-LBx needs to be evaluated in larger animalmodels such as rats, pigs, and non-human primates. Third, a fewinconsistencies were observed in plasma eGFP mRNA findings withdifferent primer sets. Although eGFP mRNA measured with both primer setswas overall highly correlated (r=0.93, root mean square difference=1.85cycle threshold), the difference between plasma eGFP mRNA levels of micetreated 0.59 MPa and higher PNPs were less prominent with primer set B.More robust and reproducible measurements of RNA levels with dropletdigital PCR³⁷ could mitigate these issues and enable reliable detectionof small differences in FUS-LBx.

Conclusions

This study showed that an acoustic pressure as low as 0.59 MPa wassufficient for FUS-LBx. Although higher peak negative acoustic pressurestended to be associated with a better yield of liquid biopsy, it wasalso associated with a higher burden of microhemorrhage. MRI can be usedin not only guiding the FUS targeting of a specific brain region butalso providing predictions of the biomarker release level and potentialhemorrhage extent. Future studies are warranted to develop MRI-guidedFUS-LBx to improve its safety and efficacy.

Methods

FUS-LBx Treatment Procedure

All animal studies were reviewed and approved by the InstitutionalAnimal Care and Use Committee of Washington University in St. Louis inaccordance with the National Institutes of Health Guidelines for animalresearch.

Mice (Strain 550, 6-8 weeks, n=20, Charles River Laboratory, Wilmington,Mass., USA) were implanted with GL261 murine glioblastoma cells on day 0using an established protocol¹⁵. The growth of the tumor was monitoredusing a clinical MRI scanner (Ingenia 1.5T, Philips Healthcare, Best,the Netherlands) coupled with a small animal coil (FUS Instruments Inc.,Toronto, Ontario, Canada) as shown in FIG. 5 twice per week. Once themaximum diameter of the tumor reached 2 mm as measured based on thecontrast-enhanced MRI, the mice were randomly assigned into four groups:control group that received no treatment (n=5) and three groups thatreceived FUS treatment with three different PNP: 0.59 MPa (n=5), 1.29MPa (n=5), and 1.58 MPa (n=5). All other parameters were the same acrossthe treatment groups: 1.44 MHz, sonication duration: 240 s; treatmenttarget: 1 (at the center of the tumor); pulse repetition frequency: 1Hz; duty cycle: 1%; pulse length: 10 ms. All FUS sonications wereperformed on post-implantation days 15 or 16.

The details of the sonication procedure and the dose and concentrationof the microbubbles have been described previously¹⁵. Briefly, the micewere treated by FUS using a clinical MR-guided FUS system (Sonalleve V2,Profound Medical Inc., Mississauga, Canada) that integrated a clinicalMRI scanner (Ingenia 1.5T, Philips Healthcare, Best, the Netherlands)with a 256-element phased array FUS transducer. The system wasconfigured for small animal study by coupling with a small animaladaptor (FUS Instruments Inc., Toronto, Ontario, Canada) placed abovethe acoustic window of the FUS transducer (see e.g., FIG. 5A and FIG.5B). The adaptor consisted of a small animal coil, a standoff, and amouse bed. The software of the MR-guided FUS system was modified toachieve tumor targeting under the guidance of MR images of mouse brainsacquired using the small animal coil. It was also modified to performpulsed FUS sonication at low-pressure levels used in this study by onlyusing 128 elements. The acoustic fields generated by the phased arraytransducer was calibrated by a fiber optic hydrophone using previouslypublished methods (see e.g., FIG. 5C)^(22,38). The full width at halfmaximum of the axial beam and lateral beam was 12.10 mm and 1.37 mm,respectively.

Contrast-enhanced T1-weighted turbo spin-echo MR images (TR, 500 ms; TE,13 ms; acquisition matrix, 96×96; resolution, 0.2 mm×0.2 mm×0.5 mm) wereacquired before and after the FUS treatment to quantify changes inintratumoral and peritumoral enhancement.

Plasma eGFP mRNA Level Quantification

Blood samples of 500-800 μL were collected from the heart about 20 minafter FUS sonication and prepared for qPCR analysis of eGFP mRNA. Themethods of qPCR analysis of eGFP mRNA have been described in Example 1.Briefly, RNA was extracted from the plasma samples using miRNeasyserum/plasma kit (Catalog no. 217184, Qiagen, USA) followed by AgencourtRNAClean XP beads (Catalog no. A63987, Beckman Coulter Inc., USA).Extracted RNA was then converted to cDNA using the Applied Biosystemshigh-capacity cDNA reverse transcription kit (Catalog no. 4368814, TermoFisher Scientifc, USA). Two primer sets were used to quantify eGFPlevels. 5.8S rRNA was used as the internal control to normalize the PCRdata by calculating cycle threshold change (ΔC_(T)) by subtracting C_(T)of the eGFP (C_(T,eGFP)) by the C_(T) of the housekeeping gene, 5.8srRNA (C_(T,5.8S)). The relative gene expression level was determinedusing the 2^(−ΔCT) method: 2^(−ΔC) ^(T) =2^(−(C) ^(T-eGFP) ^(−C)^(T,5.8s) ⁾.

MRI Image Analysis

MR image processing and analysis were performed using tools available inFSL v5.0.10³⁶. First, tumor regions were segmented semi-automatically onthe pre-sonication contrast-enhanced T1-weighted images using ITK-SNAPv3.6.0³⁵. Second, on each pre-sonication contrast-enhanced T1-weightedMRI image of the brain tumor, a spherical control mask with 1 mm radiuswas drawn in the normal appearing brain and its mean and standarddeviation were calculated. Third, all contrast-enhanced T1-weightedimages (both pre-sonication and post-sonication images) were intensitynormalized by subtracting the mean and then dividing by the standarddeviation of the signal intensity within the control mask. Fourth, theperitumoral area was defined as the brain regions within 2 mm vicinityof the tumor. A preliminary mask was created using the distancemapcommand in FSL. This mask was then edited manually to exclude voxelsoutside the brain parenchyma. Finally, to quantify post-sonicationchanges in MRI contrast enhancement, the difference between normalizedpost-sonication and pre-sonication T1-weighted images was calculatedwithin the intratumoral and peritumoral area masks.

Histologic Analysis

After blood collection, all mice were transcardially perfused with 0.01M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde.Brains were harvested and prepared for paraffin sectioning. The mousebrains were horizontally sectioned to 15 μm slices and used for H&Estaining. Whole slide images of H&E tissue sections were digitallyacquired using Nanozoomer 2.0-HT slide scanner (Hamamatsu Photonics,Hamamatsu City, Japan). For each mouse, the H&E stained slice with thelargest tumor surface area and least artifact was selected forquantitative analysis. QuPath v0.1.3³⁹ was used to detect areas ofmicrohemorrhage. After color deconvolution (hematoxylin vs. eosin),areas of microhemorrhage was detected using the positive-pixel countalgorithm. Microhemorrhage density was calculated as the percentage ofmicrohemorrhage surface area over the entire evaluated surface area (%).

Statistical Analysis

All statistical analyses were performed using R statistical softwarev3.5.0 (https://www.R-project.org/)⁴⁰ and graphically displayed usingggplot2 package v2.2.1⁴¹. For group comparisons, the non-parametricMann-Whitney U test was used. Associations between two continuousvariables were assessed using Pearson correlation analysis. eGFP plasmalevels were log transformed for correlation analysis. Statisticalsignificance was set at p<0.05.

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Example 3: Noninvasive and Localized Blood-Brain Barrier Opening andDetection of Brain-Specific Biomarkers

This example shows the pressure field measurements of the designedtransducer and the evaluation of its performance and the detection ofbrain-specific biomarkers.

Blood-brain barrier (BBB) opening with focused ultrasound andmicrobubbles (MB) have been demonstrated to be a noninvasive, targeted,safe, and effective technique mostly in anesthetized animal models.However, the requirement of anesthesia can limit the future clinicalapplication of this noninvasive technology for the treatment of braindiseases as patient real-time feedback is critical to ensure the safetyof the treatment. So far, only one study demonstrated the feasibility ofBBB opening using FUS in awake non-human primates after training thenon-human primates to cooperate during the treatment. No study hasevaluated the feasibility of FUS-induced BBB opening in the mostcommonly used animal model—mice, because of the technical challengesassociated with designing a device for FUS sonication in awake mice.

The blood-brain barrier (BBB) blocks large molecules (>400 Da) fromentering the central nervous system (CNS). Focused Ultrasound (FUS)combined with microbubble is the only available technique tonon-invasively, locally, and transiently open the BBB. Previouspreclinical research using small animal models has demonstrated thistechnique as a promising way to treat various brain disorders. Thistechnique has also been tested on non-human primates with success.Clinical Studies with FUS are actively investigated for treatment ofbrain tumor and Alzheimer's disease. Besides its clinical translationpotential, this technique has a broad application in preclinicalresearch. It can be used to delivery various agents to the brain, suchas chemo drugs, proteins, peptides, nanoparticles, gene vectors, andstem cells. However, currently there is a lack of devices that areaffordable, high throughput, and dedicated for small animal research.Here is disclosed a new FUS-induced BBB opening device that meets theseneeds. The transducer was made using an inexpensive flat ultrasoundtransducer coupled to a 3D printed acoustic lens. The cost of thetransducer was about $80, while the FUS transducer commonly used inpreclinical research can cost over $2,000. Multiple transducers can beused simultaneously to achieve high throughput treatment of multiplemice at the same time.

A wearable helmet was designed for non-invasive, targeted FUS sonicationof the mouse brain while the animals were awake. The helmet had amodular design featuring easy removal and installing of the unit fortargeting different brain regions. The performance of the helmet ininducing BBB opening at the caudate putamen was assessed in four awakemice and four anesthetized mice in the presence of intravenouslyinjected microbubbles. Evan's blue was co-injected with the microbubblesfor the evaluation of the BBB permeability using fluorescence imaging ofex vivo brain slices.

The whole helmet with ultrasound transducer and wirings weighed 6.6 g.The constraint design of the helmet minimized the effect of mousemovement on targeting. The helmet achieved localized BBB opening at thetargeted brain location. The fluorescence intensity of the Evan's bluein the brains of awake mice was higher than that in anesthetized mice,suggesting that FUS-induced BBB opening was affected by anesthesia.

The helmet design of the FUS device provides an innovative tool to studyFUS-induced BBB opening in awake mice.

FIG. 9 is a photo of the transducer. FIG. 10 is a photo of a mousetreated using the device. FIG. 11 is a photo showing the successful drugdelivery to the mouse brain, indicated by the leakage of the blue color.FIG. 12 is an image of spatially precise drug delivery to thehippocampus, indicated by the enhanced fluorescence observed on theright side of the of the mouse hippocampus.

An MRgFUS system was also designed for a pig (see e.g., FIG. 13). Thisdesign can be easily configured for clinical use by humans. The MRgFUSsystem is designed to integrate into the bore of an MRI scanner (seee.g., FIG. 13C).

The system allows for planning and targeting of image-guided focusedultrasound liquid biopsy (FUS-LBx) (see e.g., FIG. 14); monitoring andfeedback control using an US sensor to monitor microbubble response toUS (see e.g., FIG. 15); and outcome assessment (see e.g., FIG. 16).

Described herein is also a method of monitoring cavitation (e.g.,microbubble behavior) (see e.g., FIG. 15). The FUS system comprised asensor that measures acoustic signal. This monitoring informs if thepressure should be increased or decreased by measuring the acousticsignal. Here, the IC line (blue) monitors the bubbles and the SC showsthe active calibration to achieve the amount of pressure delivery. Thiscan be used to monitor for safety.

Another routine MRI is performed to detect the leakiness of the areathat was administered US.

FUS-LBx was performed in a large animal model (pig). FIG. 13 shows animage of the pig MRgFUS system. Brain-specific biomarkers, glialfibrillary acidic protein (GFAP), myelin basic protein (MBP), and S100calcium-binding protein B (S100B) were measured before and after FUS.Increased levels of the brain specific biomarkers were shown inincreased levels post-FUS (see e.g., FIG. 17).

What is claimed is:
 1. A method of non-invasively releasing biomarkersfrom a brain or a brain region across a blood brain barrier (BBB) of asubject comprising: (i) applying a focused ultrasound (FUS) to the brainor the brain region, wherein the FUS is applied for a period of time andat an acoustic pressure sufficient to disrupt the BBB and release adetectable quantity of a biomarker across the BBB; (ii) obtaining abiological sample from the subject after applying the FUS to a subjectbrain or a subject brain region; and (iii) detecting a biomarker in thebiological sample.
 2. The method of claim 1, wherein the biologicalsample is a biological fluid selected from the group consisting of:blood, cerebral spinal fluid (CSF), interstitial fluid (ISF), serum, andplasma.
 3. The method of claim 1, wherein the period of time sufficientto disrupt the blood brain barrier (BBB) and release a detectablequantity of a biomarker across the BBB is between about 1 minute andabout 4 minutes; or the acoustic pressure sufficient to disrupt theblood brain barrier (BBB) and release a detectable quantity of abiomarker across the BBB is between about 0.1 MPa and about 10 MPa. 4.The method of claim 1, wherein the biomarker comprises genetic material.5. The method of claim 4, wherein the genetic material is selected fromcell-free RNA, cell-free DNA, mRNA, circulating tumor DNA (ctDNA), orplasma DNA concentration.
 6. The method of claim 1, wherein thebiomarker is selected from D-2-hydroxyglutarate (D2HG) or IDH1(R132H)mutation.
 7. The method of claim 1, wherein the method comprises:scanning a subject head using a magnetic resonance imaging (MRI) scannerand stereotactically coregistering the subject head and identifying aregion to be targeted with the FUS.
 8. The method of claim 1, comprisingassessing the effectiveness of the BBB disruption or release ofbiomarkers comprising measuring MRI contrast before and after FUS,wherein an increase in MRI contrast after FUS compared to the MRIcontrast before FUS indicates successful release of biomarker from thebrain or brain region.
 9. The method of claim 1, wherein detecting thebiomarker in a biological sample comprises genetic testing orsequencing.
 10. The method of claim 1, comprising: extracting cell-freeor exosomic DNA or RNA from the biological sample; and detecting somaticmutations or somatic variants in the DNA or RNA using a targetedultra-deep sequencing technology selected from the group consisting of:ddPCR, AmpliSeq, and HaloPlex sequencing.
 11. The method of claim 1,comprising comparing a level of a biomarker in the biological sampleafter administering the FUS to a biological sample obtained from thesubject before FUS or of a matched control sample or standard sample.12. The method of claim 1, wherein the brain or brain region comprises atumor, lesion, or suspected tumor; or the subject has or is suspected ofhaving a central nervous system cancer or tumor; a brain tumor, a brainlesion, a neurological disease, disorder, or condition, or aneurodegenerative disease disorder, or condition.
 13. The method ofclaim 1, wherein the FUS is applied for a period of time and at apressure sufficient to rupture cells to release biomarkers.
 14. Themethod of claim 1, comprising administering microbubbles to a subject inan amount sufficient to disrupt the BBB upon application of FUS.
 15. Themethod of claim 1, comprising providing an acoustic sensor; anddetecting an acoustic signal, wherein the sensor is capable of measuringor monitoring cavitational acoustic emissions.
 16. A system suitable foruse in delivering focused ultrasound (FUS) to a brain or a region of abrain of a subject, comprising: a device comprising a plurality ofmodular pieces configured to deliver ultrasound to the brain or a brainregion; an ultrasound transducer configured to emit a FUS beam; and anacoustic lens configured to control the FUS beam direction.
 17. Thesystem of claim 16, wherein the system is configured for incorporationinto a magnetic resonance imaging (MRI) scanner, wherein the MRI scanneris configured to provide an MRI image for guiding or monitoring of theFUS.
 18. The system of claim 16, wherein the system comprises a helmetconfigured to fit over a head of a subject; and the helmet isdemountably coupled to the ultrasound transducer; the ultrasoundtransducer is demountably coupled to the acoustic lens; the ultrasoundtransducer is a flat ultrasound transducer; the acoustic lens is a 3Dprinted acoustic lens; or the acoustic lens is a convex lens.
 19. Thesystem of claim 16, wherein the system is suitable for use in parallelwith a plurality of the systems.
 20. The system of claim 16, comprisinga sensor capable of measuring or monitoring cavitational acousticemissions.
 21. The system of claim 16, wherein the ultrasound transduceris configured to emit an acoustic pressure of less than about 10 MPa.22. The system of claim 16, wherein the system comprises one to eighttransducers.